mechanism and activation for allosteric adenosine 5 ...at secondary centers usually exhibits isotope...

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 by The American Society of Biological Chemists, Inc.

Vol. 261, No. 10, Issue of April 5, pp. 4451-4459,1986 Printed in U.S.A.

Mechanism and Activation for Allosteric Adenosine 5’-Monophosphate Nucleosidase KINETIC a-DEUTERIUM ISOTOPE EFFECTS FOR THE ENZYME-CATALYZED HYDROLYSIS OF ADENOSINE 5”MONOPHOSPHATE AND NICOTINAMIDE MONONUCLEOTIDE*

(Received for publication, July 15, 1985)

Mark T. SkoogS From the Department of Chemistry, Indiana University, Bloomington, Indiana 47405

The kinetic a-deuterium isotope effect on VmJKm for hydrolysis of NMN catalyzed by AMP nucleosidase at saturating concentrations of the allosteric activator MgATP2- is kH/kD = 1.155 f 0.012. This value is close to that reported previously for the nonenzymatic hydrolysis of nucleosides of related structure, suggesting that the full intrinsic isotope effect for enzymatic NMN hydrolysis is expressed under these conditions; that is, bond-changing reactions are largely or completely rate-determining and the transition state has marked oxocarbonium ion character. The kinetic a-deuterium isotope effect for this reaction is unchanged when deuterium oxide replaces water as solvent, corroborating this conclusion. Furthermore, this isotope effect is independent of pH over the range 6.95-9.25, for which values of V,,JKm change by a factor of 90, suggesting that the isotope-sensitive and pH-sensitive steps for AMP-nucleosidase-catalyzed NMN hydrolysis are the same.

Values of kH/kD for AMP nucleosidase-catalyzed hydrolysis of NMN decrease with decreasing saturation of enzyme with MgATP2- and reach unity when the enzyme is less than half-saturated with this activator. This requires that the rate-determining step changes from cleavage of the covalent C-N bond to one which is isotope-independent.

In contrast to the case for NMN hydrolysis, AMP nucleosidase-catalyzed hydrolysis of AMP at saturating concentrations of MgATP2- shows a kinetic a-deuterium isotope effect of unity. Thus, covalent bond- changing reactions are largely or completely rate-determining for hydrolysis of a poor substrate, NMN, but make little or no contribution to rate-determining step for hydrolysis of a good substrate, AMP, by maximally activated enzyme. This behavior has several prece- dents.

Adenosine 5‘-monophosphate nucleosidase (EC 3.2.2.4) from Azotobacter vinelandii catalyzes the hydrolysis of 5’- AMP to adenine and ribose 5-phosphate (1-3). The enzyme is a hexamer of apparently identical subunits that contain six sites that bind the allosteric activator, MgATP2-, and three sites that bind substrate and substrate analogs (4). Although

* This work was supported by Research Grant PCM 77-00171 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement”, in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$’ Current address: Merrell Dow Research Institute, 2110 E. Gal- braith Rd., Cincinnati, OH 45215.

AMP nucleosidase is specific for AMP and a few of its analogs, NMN is a poor substrate (5). The V,, for AMP hydrolysis is 34 IU/mg of protein and that for NMN hydrolysis 0.8 IU/mg of protein. The K,,, for AMP hydrolysis is 0.12 mM and that for NMN hydrolysis is 8 mM (5). Activation by MgATP2- increases the rate of substrate hydrolysis but not the binding constants for substrate analogs (4). The allosteric inhibitor of AMP nucleosidase, HPOq-, binds to six interdependent sites that are identical with or that cause mutual exclusion of MgATP2- from the activator sites (4). Inactivation with phosphate results in dissociation of the hexameric enzyme to inactive dimers (6).

Under optimal conditions the turnover number for AMP nucleosidase-catalyzed hydrolysis of 5‘-AMP is 60 s-l (7). There is no evidence for enzymatic functional groups that participate as nucleophiles in the bond-cleavage step or stabilize the oxocarbonium ion, resulting from unimolecular decomposition of the N-glycosidic bond at the active site. The presence of 14CH30H in the assay mixture does not result in the production of 14C-labeled methylribofuranoside 5-phosphate (8), suggesting that 5‘-AMP or an oxocarbonium ion derived from it is attacked by water bound to AMP nucleosidase at a site inaccessible to methanol.

MnATP2- binds to two classes of activator sites (9). Three high affinity MnATP2- sites have increased affinity for MnATP2- in the presence of formycin 5’-monophosphate, an AMP analog. Three low affinity sites have decreased affinity for MnATP2- in the presence of formycin 5‘-monophosphate. Assuming that MgATP2- and 5’-AMP interact with the enzyme in a manner analogous to that of MnATP2- and formycin 5’-monophosphate, it has been proposed that there are six sites on AMP nucleosidase that can bind either substrate or modifier. Binding of substrate to a site would make the neighboring sites prefer to bind activator, and vice versa, resulting in two classes of activator sites in the presence of substrate (9). Experimental data are also consistent with an enzyme that has three substrate sites distinct from six activator sites.

The equilibrium a-deuterium isotope effect for hydration of acetaldehyde is k&, = 1.37 (10). The primary origin of this isotope effect is the increased looseness of out-of-plane bending vibrations for hydrogen attached to trigonally hybridized carbon compared to hydrogen attached to tetrahe- drally hybridized carbon (11). This calculation suggests that k H / k D = 1.4 may be the upper limit for kinetic a-secondary deuterium isotope effects for the unimolecular decomposition of tetrahedral carbon centers as the transition state becomes progressively more product-like. One of the largest kinetic 01-

deuterium isotope effects observed for S,1 solvolysis is that for the solvolysis of arenesulfonates, kH/kD = 1.24 (11).

445 1

4452 Activation of A M P Nucleosidase-catalyzed Hydrolysis of A M P and N M N

Smaller than maximum observed kinetic a-deuterium isotope effects for S,1 reactions are attributed to interference of the leaving group or nucleophile with the bending vibrations of the a-hydrogen. The limiting case of such interference is the S,2 mechanism. Here the motions of the leaving group and nucleophile have approximately opposite effects on the vibrations of the a-hydrogen, resulting in values of kH/kD near unity. Values of kH/kD between 0.96 and 1.04 have been observed for a variety of nucleophilic displacement reactions at primary carbon centers (11-17). Nucleophilic displacement at secondary centers usually exhibits isotope effects 4% larger than those for analogous reactions at primary carbon centers (11).

The interpretation of isotope effects on enzyme-catalyzed reactions is complicated by the complex multistep nature of enzyme mechanisms. The measured kinetic constants, V,,,, K,, and V,,/K,, are complex expressions of rate constants for elementary steps. If steps other than those that are isotope-sensitive are rate-determining, the isotope effects on these constants will tend to unity. Useful strategies for the observation of the isotope effect on the bond-changing step of an enzyme-catalyzed reaction include comparison of deuterium and tritium isotope effects on V,,,/K, (18, 19), variation of the concentrations of other substrates (20,21), choos- ing a different source of enzyme (20,22), varying pH (19,20, 22-25) or ionic strength (19, 22, 23), and use of alternate substrates (19-23, 26, 27). Competitive determinations of isotope effects on enzyme-catalyzed reactions yield the isotope effect on V,,,/K, (28-31). In this study competitive determinations using the double label technique (32,33) were used to study the kinetic cy-secondary deuterium isotope effect on V,,,/K, for the AMP nucleosidase-catalyzed hydrolysis of AMP and NMN as a function of the concentration of the allosteric activator, MgATP2-, and for enzyme-catalyzed NMN hydrolysis as a function of pH.

EXPERIMENTAL PROCEDURES

Materials [carb~nyZ-'~CC]Nicotinamide, [4-3H]NAD, ['4C]toluene liquid scin-

tillation counting standards, and Bray's solution were products of New England Nuclear. [2-3H]AMP, [8-14C]AMP, and [8-14C]adenine were products of Amersham/Searle. [1-'HIInosine and [l'-'H]NAD were generous gifts of Dr. H. G. Bull of Merck Sharp & Dohme Research Laboratories, Rahway, NJ. Sephadex G-10 was obtained from Pharmacia Fine Chemicals. AG 1-X4 and AG 50-X8 ion exchange resins were obtained from Bio-Rad. A purified preparation of pig brain NAD glycohydrolase was supplied by M. Sankovic of Indi- ana University. Nucleotide pyrophosphatase from Crotalus adaman- teus venom was obtained from Sigma. A preparation of AMP nucleosidase >95% homogeneous and at a concentration of 100 IU/ml was prepared by the methods of Schramm and Leung (34). All other reagents were the finest commercial grades available and were used without further purification unless indicated otherwise in the. text. Glass distilled water was used throughout.

Methods Purification and Assay of Nucleoside Phosphotransferase from Car-

rots-This activity was purified from carrots purchased at a local grocer through the third step, ammonium sulfate fractionation, of the preparative procedure of Brunngraber and Chargaff (35) and Brun- ngraber (36). This preparation contained 2'-nucleotidase, 3'-nucleotidase, and 5'-nucleotidase activities in addition to the nucleoside phosphotransferase activity. The final preparation contained 2.0 IU of activity for the synthesis of AMP from adenosine in 50 ml of enzyme solution and a specific activity of 0.018 IU/mg protein.

The preparation was assayed for the production of AMP from adenosine and phenyl phosphate. A stock solution of 40 mM adenosine, 200 mM sodium phenyl phosphate, and 200 mM sodium acetate at pH 5.0 was incubated at 37 "C. The reaction was initiated by the combination of 0.20 ml each of stock and enzyme solutions. After

incubation at 37 "C for 1 h, a 0.10-ml aliquot of the reaction mixture mixture was applied to an anion exchange column (AG 1-X4, 200- 400 mesh, acetate form, 0.5 X 3.0 cm in a Pasteur pipette, 23 "C). The column was eluted with water until the absorbance at 254 nm of the effluent returned to base-line. AMP was eluted from the column with 1 M acetic acid until the absorbance of the eluent at 254 nm returned to base-line. The amount of AMP produced was determined by the absorbance of the AMP fraction at 257 nm (37). The nucleotidase activities were assayed by following, the disappearance of nucleotide from a reaction mixture by the same method used to monitor the production of nucleotide. In this case, 0.20 ml of 5.0 mM nucleotide and 200 mM sodium acetate at pH 5.0 replaced the stock solution in the assay.

Synthesis of [8-14C,1 '-2H/5'-AMP-[8-'4C,1'-2H]Aden~~ine was prepared from [l'-*H]inosine and [8-14C]adenine by a modification of the method of Stein and co-workers (38) and Stein (39). Purine nucleoside phosphorylase was purified from Escherichia coli and assayed by the methods of Stein and co-workers (38) and Stein (39). This enzyme catalyzed base exchange in a reaction mixture consisting of 0.20 mM [l'-'H]inosine, 0.20 mM (16 pci) [8-14C]adenine, 2.2 X lo-' M KH'PO,, 15 mM Tris-HC1 and 0.050 IU of purine nucleoside phosphorylase at pH 7.5 in 1.35 ml. After incubation for 25 h at 23 "C, the reaction was quenched by immersion of the reaction vessel in boiling water for 15 min. Precipitate was removed by centrifugation at 1000 X g until the solution was clear. The components of the reaction mixture were separated by chromatography on Sephadex G- 10 (1.0 X 117 cm, 4 "C, flowing 230 ml/day with water as eluent). The elution of nucleosides and bases was monitored by their absorbance at 254 nm. Inosine eluted 81 ml past the point of application followed by hypoxanthine at 128 ml, adenosine at 270 ml, and adenine at 547 ml. The yield of [8-'4C,1'-2H]adenosine was 84% based on counts/ min.

This adenosine was converted to AMP by a small scale version of the method of Yoshikawa and co-workers (40,41). The [8-14C,1'-2H] adenosine plus 40 pmol of unlabeled adenosine were lyophilized to dryness in a 1-ml conical vial. Water was removed from the reaction vessel by the repeated addition and evaporation under dry nitrogen of absolute ethanol. A stirring bar and 0.10 ml of triethyl phosphate, distilled and stored in a dessicator at -20 "C, were added to the vial. The vial was placed in a jar also containing dessicant, the jar was sealed, and stirring was initiated. The apparatus was already at 4 "C,

"aH po:a" OH OH OH OH

( 2 - 3 1 i , l ~ ~ A M P (S""C,IL~FDAMP

SCHEME 2

Activation of AMP Nucleosidase-catalyzed Hydrolysis of AMP and NMN 4453

and 30 min were allowed for temperature equilibration of the reaction vessel. Then 7.3 pl of POCh, which had previuosly been distilled and stored dessicated at -20 "C, was added and the jar was resealed. The reaction continued for 6 h at 4 "C with continuous stirring. Excess POC13 was consumed by addition of several drops of water, and the contents of the reaction vessel were layered on the bed of a column of Sephadex G-10 (1.5 X 120 cm) and eluted with M acetic acid, 23 "C. All nucleotide products., 2',5'-ADP, 3',5'-ADP, 2'-AMP, 3'- AMP (42), and 5'-AMP, eluted as a single peak at 60 ml past the point of application and were separated from triethyl phosphate. The nucleotide fractions, as determined by absorbance at 264 nm, were applied to an anion exchange column (AG 1 X4, 200-400 mesh, acetate form, 0.5 X 1.0 cm in a Pasteur pipette, 23 "C). The monophosphates were eluted with 1.0 M acetic acid until the eluent had negligible absorbance at 254 nm. These monophosphates were then desalted by another run through the column of Sephadex G-10. The yield of the phosphorylation step for the production of a mixture of adenosine monophosphates was 38% based on counts/min of "c. This mixture was lyophilized to dryness in a reaction vessel.

Rather than remove the 2'-AMP and 3'-AMP from the 5'-AMP with the corresponding loss of yield, the mixture of adenosine monophosphates was converted to 5'-AMP by the action of a crude preparation of nucleoside phosphotransferase from carrots (35, 36). This activity transfers phosphate from nucleoside monophosphates and phenyl phosphate to nucleosides. The presence of 2'- and 3'- nucleotidase activities results in the net production of 5'-AMP when a mixture of adenosine monophosphates is incubated with a large excess of phosphate donor in the presence of nucleoside phosphotransferase. The mixture of adenosine monophosphates produced by organic phosphorylation was dissolved in 0.50 ml of 0.20 M phenyl phosphate, 0.20 M acetic acid, pH 5.00. After incubation at 37 "C for 30 min, the reaction was started by the addition of 1.5 ml of the crude nucleoside phosphotransferase preparation. The reaction was stopped after 6.5 h of incubation at 37 "C by application of the reaction mixture to an anion exchange column (AG 1-X4, 200-400 mesh, acetate form, 0.5 X 3.0 cm in a Pasteur pipette a t 23 "C). The column was eluted with water until the absorbance at 254 nm of the effluent had returned to base-line. The AMP was eluted with 1.0 M acetic acid and desalted by chromatography on Sephadex G-10. The yield of the enzymatic reaction based on counts/min of 14C was greater than 98%.

The purity of this preparation of 5'-AMP was assayed by proton NMR spectroscopy and through the specificity of AMP nucleosidase. A Varian HR-220 NMR spectrometer was used to obtain the proton NMR spectrum of the 5'-AMP preparation and commercial 2'-AMP, 3'-AMP, 5'-AMP, 2',5'-ADP, and 3',5'-ADP in D20. The spectra of the prepared and commercial 5'-AMP are the same and are clearly different from the spectra of the other compounds. However, resolu- tion was insufficient to detect trace contaminants. AMP nucleosidase catalyzes the hydrolysis of adenosine, 2'-AMP, or 3'-AMP at less than of the rate of hydrolysis of 5'-AMP (5). An aliquot of this 5'-AMP was incubated with AMP nucleosidase in the presence of 1 mM MgATP2- at pH 8.00 and 30 "C. Separation of adenine from AMP resulted in the recovery of no detectable AMP, as determined by monitoring chromatographic fractions for absorbance at 257 nm and for the presence of 14C. The recovery of adenine was quantitative. This suggests that this synthesis yielded pure [8-'4C,1'-2H]5'-AMP.

This 14C-labeled 5'-AMP was then combined with an appropriate amount of [2-3H]5'-AMP, which had previously been chromatographed on Sephadex G-10, and lyophilized to dryness in 5-ml pear- shaped flasks. These flasks had previously been immersed upright in boiling glass distilled water for 10 min to remove any traces of inorganic phosphate, an inhibitor of AMP nucleosidase. These aliquots of double-labeled 5'-AMP were stored dessicated at -20 "C until needed.

Synthesis of [4-3H]NMN and [c~rbonyl-~~C,l '-2H]NMN-[car- ~ O ~ ~ ~ - ' ~ C , ~ ' - ~ H ] N M N and [4-3H]NMN were prepared from [1'-'H] NAD and [4-3H]NAD, respectively, by a modification of the methods of Bull and co-workers (26) as outlined below.

[~arbonyl-'~C]Nicotinamide and [l'-'H]NAD were chromatographed on Sephadex G-10 (1.7 X 117 cm, flow rate of 230 ml/day with lo-' M acetic acid eluent, 4 "C) prior to use. A solution of 3.3 pmol (29 pCi) of [~arbonyl-~~C]nicotinarnide and 5.0 pmol of [l'-'H] NAD was lyophilized to dryness in a 5-ml pear-shaped flask. A reaction mixture consisting of 0.40 ml of 0.10 M potassium phosphate at pH 7.50 and 0.40 ml of a stock solution containing 0.30 IU of pig brain NAD glycohydrolase activity was added to the flask. After 4.5 h of incubation at 23 "C, the reaction was quenched by application of

the reaction mixture to a cation exchange column (AG 50W-X8,400- 800 mesh, H+ form, 0.5 X 3.0 cm in a Pasteur pipette, 23 "c). Adenosine 5"diphosphate 3"-ribose was eluted in 10 ml of 0.10 M formic acid followed by and separated from [~arbonyl-'~C,l'-'H]NAD in 57 ml. Elution with 0.10 M NH4OH resulted in the recovery of [c~rbonyl-~~C]nicotinamide in 18 ml. The elution of these species from the column was monitored by the absorbance of the effluent at 254 nm. The [~arbonyyl-'~C,l'-~H]NAD contained 30% of the radioactivity initially present in the nicotinamide and was chromatographed on Sephadex G-10 prior to further use.

The [~arbonyl-'~C,l'-~H]NAD was then combined with an appropriate amount of [4-3H]NAD, and this mixture was lyophilized to dryness in a pear-shaped flask. A solution of 0.20 ml of 0.10 M Tris acetate and 0.07 M MgClz at pH 7.4 was added to the flask, and the mixture was allowed to equilibrate a t 37 "C for 30 min. This reaction was started by the addition of an excess of nucleotide pyrophosphatase and incubation continued for another hour. The reaction was quenched by application of the reaction mixture to an anion exchange column (AG 1-X4, 200-400 mesh, acetate form, 0.5 X 2.0 cm in a Pasteur pipette, 23 "C). Elution with 20 ml of 0.010 111 acetic acid resulted in the recovery of a mixture of [~arbonyl-'~C,l'-~H]NMN and [4-3H]NMN. The NMN was desalted by chromat'ography on Sephadex G-10. The yield of NMN from NAD was 86% based on counts/min of 14C. Aliquots of the double-labeled NMN were lyophilized to dryness in pear-shaped flasks. These flasks had previously been immersed upright in boiling glass distilled water for 10 min to remove any traces of inorganic phosphate, an inhibitor of AMP nucleosidase. These aliquots of double-labeled NMN were stored dessicated at -20 "C until needed.

Isotope Effect Determinutions for AMP Nucleosidase-catalyzed Hy- drolysis of AMP-A single procedure was used for all determinations of kinetic a-deuterium isotope effects for the AMP nucleosidase- catalyzed hydrolysis of AMP in the presence of MgATP2-.

The reaction mixture was prepared in the flask of double-labeled AMP by the addition of stock solutions starting with water. After the addition of water, an aliquot of the double-labeled starting material sufficient to yield 5000 cpm of 14C was added to each of three scintillation vials. Stock solutions of other reagents were added to the flask, resulting in a reaction mixture containing double-labeled AMP, 0.10 M Tris, and the desired concentration of MgATP2- at pH 8.00 in 0.5 ml. The desired concentration of MgATPz- was obtained by the presence of MgC12 at twice the concentration of ATP. The actual concentration of MgATP2- was calculated from its stability constant of 73,000 M" (43). The stability constant of MgAMP of 100 M" (44) indicates that not more than 10% of the AMP was present as MgAMP in any experiment. The reaction mixture was incubated at 30 "C for 30 min. The reaction was started by the addition of enough AMP nucleosidase stock solution to result in the production of 15,000 cpm of "C-labeled adenine in the desired reaction time. The reaction was quenched by application of the reaction mixture to an anion exchange column (AG 1-X4, 200-400 mesh, acetate form, 0.5 X 0.5 cm in a Pasteur pipette, 23 "C). Adenine was eluted with water, and the fraction was divided equally among three scintillation vials. AMP was subsequently eluted with 1.0 M acetic acid. The AMP nucleosidase and ATP in the reaction mixture remained bound to the resin, which was discarded. Aliquots of the AMP fraction sufficient to yield 200 cpm of 14C were dispensed into each of three scintillation vials. All scintillation vials were then lyophilized to dryness prior to preparation for scintillation counting. The remaining double-labeled AMP was desalted and stored for further use.

In the absence of MgATP2-, a kinetic a-tritium isotope effect was measured by competitive determinations with [1'-3H]AMP and [5'- "CIAMP as substrates. These double-labeled compounds were prepared by enzymatic synthesis from [l-3H]ribose and [6-'4C]glucose, respectively, and purified chromatographically (45). The reaction mixtures contained 1.0 mM AMP, 0.10 M triethanolamine, and up to 1 mM MgClz at pH 8.0. After the desired extent reaction, ribose 5- phosphate was separated from AMP by chromatography on charcoal, and the isotopic composition was determined by scintillation counting (45).

Isotope Effect Determinations for the AMP Nucleosidase-catalyzed Hydrolysis of NMN-A single procedure was used for all determinations of the kinetic a-deuterium isotope effect for the AMP nucleosidase-catalyzed hydrolysis of NMN.

The reaction mixture was prepared in the flask of double-labeled NMN by the addition of stock solutions starting with water. Following the addition of water, aliquots of NMN sufficient to yield 5,000 cpm

4454 Activation of A M P Nucleosidase-catalyzed Hydrolysis of A M P and NMN of 14C were dispensed into each of three scintillation vials that were set aside. Reagents were added from stock solutions to result in 0.50 ml of a reaction mixture of composition 0.10 M buffer at the required pH and the desired concentration of MgATP2-. For pH 7.50 to 9.25, Trk was the buffer. At pH 6.95, triethanolamine was the buffer. At values of pH other than 8.00,O.lO M KC1 was added to stabilize AMP nucleosidase. The desired concentration of MgATP2- was obtained by the presence of MgClz at twice the concentration of ATP. The actual concentration of MgATP2- was calculated from its stability constant of 73,000 M" (43). If the formation constant for MgNMN is similar to that for MgAMP, 100 "' (44), or MgNAD, 38 M" (46), less than 8% of the NMN in any reaction mixture was present as MgNMN. The reaction mixture was incubated at 30 "C for 30 min before the reaction was started by the addition of enough AMP nucleosidase to yield 15,000 cpm of 14C-labeled nicotinamide in the desired reaction time. The reaction was quenched in less than 2 min by the application of the reaction mixture to an anion exchange column (AG 1-X4, 200-400 mesh, acetate form, 0.5 X 1.0 cm in a Pasteur pipette, 4 "C). The NMN and nicotinamide were eluted directly onto the bed of a column of Sephadex G-10 (1.0 X 117 cm, flow rate 230 ml/day with 0.010 M acetic acid eluent, 4 "C) in 3.5 ml of 0.010 M acetic acid. ATP and AMP nucleosidase remain bound to the ion exchange resin which was discarded after a single use. The elution of NMN and nicotinamide from the Sephadex column was monitored by absorbance of chromatographic fractions at 262.5 nm. Typically NMN eluted 44 ml past the point of application followed by nicotinamide at 112 ml. Fractions containing nicotinamide were pooled and divided equally among three scintillation vials. The NMN fractions were pooled and aliquots sufficient to yield 200 cpm of I4C were dispensed into each of three scintillation vials. All scintillation vials were lyophilized to dryness prior to scintillation counting. The remaining unreacted NMN was lyophilized to dryness in a reaction vessel to await further use.

Scintillation Counting-The scintillation mixture used in the determinations of isotope effects on the AMP nucleosidase-catalyzed hydrolysis of AMP consisted of 1.500 ml of water and 10.00 ml of Bray's solution and for NMN, 0.999 mi of water and 10.00 ml of Bray's solution. The water was dispensed with a microburet to assure consistency in the amount of this potent quencher in the mixture. The Bray's solution was measured with an automatic dispenser. Counting was done by a Packard Tri-Carb Liquid Scintillation Spec- trometer, Model 3255, under conditions and by methods described previously (20, 26). Each vial was counted for 60 min or until lo6 counts were accumulated in either channel. The vials were counted three consecutive times and the results averaged to determine the isotope effect.

Calculations-Kinetic isotope effects were calculated by comparison of the isotopic composition of reactant and product as derived for the case of a reaction first order in isotopically labeled reactants which are in intermolecular competition (47-50). R is the ratio of heavy to light isotopic species in initial reactant (Ro) and product ( l i p ) . From the data provided by the liquid scintillation spectrometer, Ro/Rp (20,51) and the isotope effects are calculated by methods used previously (20, 26, 33). When more than 10% of initial reactant is converted to product, the isotope effect was corrected for this amount of conversion (20,26,33,47). The amount of substrate converted was determined to accuracy of rt0.5%.

The equation used to calculate Ro/Rp from the radioisotopic composition of products and reactants assumes that p particles emitted by product and reactant molecules are detected with equal efficiency by the scintillation mixture and liquid scintillation spectrometer. The efficiency of detection of disintegrations from a species is the intrinsic efficiency of the detection system with respect to that species. Ro/Rp calculated from counts/min at 100% conversion ((Ro/Rp)1w%) is the ratio of intrinsic counting efficiencies for the labeled product and reactant:

where E = intrinsic counting efficiency, subscript R denotes reactant, and subscript P denotes products. The ratios of disintegrations/min cancel since after 100% conversion the reactant and product are of identical radioisotopic composition. If (Ro/Rp)l~% is unity, Ro/Rp calculated from counts/min is used to calculate isotope effects. If the

intrinsic counting efficiencies of product and reactant differ, (Ro/ Rp)lm% is not unity. Then the Ro/Rp calculated from counts/min must be divided by (RO/Rp)1m% prior to use in calculating isotope effects. An alternative method for the calculation of Ro/Rp converts counts/ min to disintegrations/min using known intrinsic counting efficiences for each labeled species. However, (Ro/Rp)lm% may be determined easily and with greater precision than an individual intrinsic counting efficiency resulting in more precise determinations of isotope effects.

of [8-'4Cc,1'-2H]AMP and [2-3H]AMP and two determinations with a The (Ro/RP)1m% calculated from two determinations with a mixture

mixture of [8-14C]AMP and [2-3H]AMP is 1.043 f 0.008. The values of Romp determined with [8-14C]AMP and [8-14C,1'-2H]AMP are identical within experimental error, suggesting that the radioisotopic purity of the deuterated, double-labeled AMP is comparable to that of the laboratory purified commercial preparations of radioisotopically labeled AMP. This value of (RO/R,)~~% was used to correct the observed values of Ro/RP

Initial Velocity of NMN Hydrolysis as a Function of the Concentra- tion of MgATP-The hydrolysis of 14C-labeled NMN was followed in a reaction mixture at 30 "c consisting of 0.5 mM NMN, 0.1 M Tris, pH 8.00, and the desired concentration of MgATP2-, which was maintained as in the isotope effect determinations. The K, for NMN hydrolysis a t saturation with MgATP2- is 7.8 mM (5), suggesting that initial velocities determined in the presence of 0.5 mM NMN will yield reasonable approximations of V,,lK,. The reaction was quenched and product determined by application of an aliquot of the reaction mixture to Whatman 3MM chromatography paper in the presence of carrier 50 mM EDTA, 10 PM formycin 5'-monophosphate, 3 mM NMN, and 3 mM nicotinamide. The chromatogram was devel- oped with isobutyric acid/concentrated NH40H/water (66:1:33). Spots of NMN and nicotinamide were cut out, immersed in scintillation mixture, and determined by scintillation counting. Enzyme- free reaction mixtures produced no 14C-labeled nicotinamide upon chromatography.

RESULTS

The kinetic secondary a-deuterium isotope effect for the AMP nucleosidase-catalyzed hydrolysis of AMP at pH 8.00 was determined as a function of the Concentration of Mg- ATP2-; isotope effects on Vmax/K, and the corresponding values of Vma.JKm are compared in Table I. The dependence of the isotope effect and V,,,,,/K, on the degree of saturation of AMP nucleosidase with MgATP2- is illustrated in Fig. 1.

TABLE I Kinetic a-deuterium isotope effects on V-/K, for the AMP

nucleosidase-catalyzed hydrolysis of AMP at pH 8.00 and 30 "C as a function of the concentration of MgATP2- Moles of

MgATPZ- Number of

of isotope effect

MgATp2- per Apparent ( V m d K h determinations mol of v t d K m b ( v m d & ) D

enzyme"

mM

0.00 0.00 0.04 1.045 f 0.010' 4 0.015 0.04 0.020 0.14 0.025 0.33 0.030 0.87 0.035 0.96 0.040 1.4 0.050 2.9 0.070 5.6 0.089 3.1 1.040 f 0.015 2 0.090 6.5 0.12 6.9 0.49 5.1 1.015 f 0.023 2 0.50 6.2 0.99 5.7 0.995 k 0.017 1 Values derived from data published in Ref. 4 and determined at

0°C. The difference in temperatures makes correlations between binding and kinetic studies inexact.

Values derived from data published in Ref. 6, calculated assuming three catalytic sites per molecule of enzyme.

e Value from Ref. 45.

Activation of A M P Nucleosidase-catalyzed Hydrolysis of A M P and NMN 4455

n x \

X I

I I I I I I 1

1.10 1.08 1.06

1.00 ,* 9' 0.98E I I 1 I I I J 1 2 3 4 5 6

Moles MgATP2-Bound Per Mole AMP Nucleosidase FIG. 1. The kinetic cy-deuterium isotope effect on V,,,.dKm

(D) and V-/K,,, (values derived from data published in Ref. 7) (0) for the AMP nucleosidase-catalyzed hydrolysis of AMP at pH 8.00 and 30 O C as a function of saturation of enzyme wit,h MgATP2- (values derived from data published in> Ref. 4). The lines were drawn by eye to fit the points with the assumption that the isotope effect approaches unity with saturation of enzyme with activator.

TABLE I1 Kinetic a-deuterium isotope effect for the AMP nucleosidase-

catalyzed hydrolysis of NMN at pH 8.00 and 30 "C as a function of the concentration of MgATP2-

Moles of MgATPZ- Number of

of isotope effect

MgATPZ- bound per V-/Kmb - mole of V - / K ~ D enzvme"

( V m d K m ) ~ determinations

mM "1 s-l

0.02 <2 0.0406 0.6, 7.6 1.017 f 0.014 2 0.0890 3 . j 48 1.017 f 0.012 2 0.183 3.6 110 0.487 5.1 160 1.042 f 0.017 4 0.587 160 1.099 f 0.012 2 0.687 5.4 160 1.128 f 0.009 2 0.987 5.7 160 1.156 f 0.026 3 1.99 6.0 180 1.154 +. 0.013 2

a Values derived from data published in Ref. 4 and determined at 0 "C. The difference in temperature makes correlations between binding and kinetic studies inexact.

* Calculated assuming three catalytic sites per molecule of enzyme.

The isotope effect is independent of the extent of binding of MgATP2- to AMP nucleosidase until the enzyme is more than 50% saturated with this ligand. At higher levels of saturation, the isotope effect decreases with increasing concentration of activator to k & ~ = 0.995 & 0.017 at 1 mM MgATP2-. The decrease observed in the isotope effect is not definitely outside of experimental error, but the trend is clearly visible in Fig. 1.

Table I1 summarizes values of Vm,,/Km and the kinetic a- deuterium isotope effect on Vm,/Km for the AMP nucleosidase-catalyzed hydrolysis of NMN at pH 8.00 as a function of the concentration of MgATP2-. To avoid problems caused by low velocity, V,, = 0.34 IU/mg protein, and high K,, 8 mM, for AMP nucleosidase-catalyzed NMN hydrolysis (5), V,,/K, was approximated from the initial velocity of NMN hydrolysis in the presence of 0.5 mM NMN. This concentration is 6% of K , for NMN hydrolysis in the presence of 1 mM MgATP2-, suggesting that this is a reasonable approximation. For the approximation to be valid under other conditions, K, must not be less than 7 mM at other concentrations of MgATP2-. Increasing concentrations of MgATP2- decrease K, for AMP nucleosidase-catalyzed hydrolysis of AMP (7) ,

suggesting that K, for NMN hydrolysis will also decrease with increasing activation of the enzyme. The dependence of the isotope effect and Vma,/Km on the degree of saturation of AMP nucleosidase with MgATP2- is illustrated in Fig. 2. The isotope effect is independent of the extent of binding of MgATP2- to AMP nucleosidase until the enzyme is more than 50% saturated with this ligand. At higher levels of saturation, the isotope effect increases with increasing concentration of activator to a constant value of k ~ / k ~ = 1.155 k 0.012, attained at a concentration of MgATP2- of 1 mM, VmaJ K , is less than 26% of its maximum value when the enzyme is half-saturated with MgATP2- and then increases until at greater than 80% saturation with activator Vma,/Km is con-

The kinetic secondary a-deuterium isotope effect for the AMP nucleosidase-catalyzed hydrolysis of NMN in the presence of 1 mM MgATP2-, necessary to stabilize the enzyme at extremes of pH, was determined as a function of pH; isotope effects on Vma,/K, and the corresponding values of V,.,/K, are collected in Table 111. The pH-independent isotope effect is k H / k D = 1.147 k 0.017.

The effect of the concentration of MgCl, on the kinetic a- deuterium isotope effect for the AMP nucleosidase-catalyzed hydrolysis of NMN at pH 8.00 is detailed in Table IV. The

stant at 160 M-' s-'.

160

120

80

40

Y E

,> e >

ATP per AMP Nucleosidase FIG. 2. The kinetic cy-deuterium isotope effect on VmJKm

(D) and values of VmPJKm (values derived from data published in Ref. 7) (X) for the AMP nucleosidase-catalyzed hydrolysis of NMN at pH 8.00 and 30 'C as a function of saturation of enzyme with MgATPZ- (values derived from data published in Ref. 4).

TABLE I11 Values of V-/K,,, and the kinetic a-deuterium isotope effects on

VJK,,, for the AMP nucleosidase-catalyzed hydrolysis of NMN at 30 "C in the presence of 0.987 mM MgATP2- as a function of OH

PH

6.50 6.90 6.95 7.50 8.00 8.10 8.60 9.10 9.25

6.95-9.25

M-' s-1

2 110

170

180 110 21

(V-lKm)n Number of

determinations (V-/K,)D of isotope

effect

1.141 f 0.015 3 1.139 rtr 0.007 2 1.156 f 0.026 3

1.15 f 0.02 3 1.147 rt 0.017 11 - ~~

V. L. Schramm, unpublished experiments. Calculated assuming three catalytic sites per molecule of enzyme.

4456 Activation of AMP Nucleosidase-catalyzed Hydrolysis of AMP and NMN TABLE IV

Kinetic @-deuterium isotope effects for the A M P nucleosidase- catalyzed hydrolysis of N M N a t p H 8.00 as a function of the

concentration of Mg2+ Number of

of isotope effect

MgATP2- ATP- MgCll kHlkD determinations

m M m M m M

0.0993 0.100 1.90 1.056 +. 0.010, 2

1.99 2.00 2.01 1.154 f 0.010 2 0.0890 0.100 0.011 1.017 f 0.010 2

TABLE V Kinetic'cu-deuterium isotope effects for the AMP nucleosidase-

catalyzed hydrolysis of N M N ut pH 8.00 in the presence of 0.987 mM MgATP2- as a function of per cent conversion of N M N

NMN converted W R P knlb

%

85 1.156 f 0.026

1.048 f 0.015 100

1.17 f 0.05 1.006 f 0.012

0.3-9.5 1.156 f 0.026

effect of MgC12 is not nearly large enough to account for the effect of increasing the concentration of MgATP2-, which also increases the concentration of MgC12, on the isotope effect.

The kinetic a-deuterium isotope effect on AMP nucleosidase-catalyzed hydrolysis of NMN at pH 8.00, 30 "C, in the presence of 1 mM MgATP2- with water as solvent is kH/kD = 1.156 +. 0.021. The isotope effect measured under the same conditions but in deuterium oxide as solvent was not detectably different from this value.

The isotope effect on the AMP nucleosidase-catalyzed hydrolysis of NMN at pH 8.00, 1 mM MgATP2- as a function of per cent conversion of NMN, is summarized in Table V. The observed isotope effect at 85% conversion extrapolates to kH/kD = 1.17 k 0.05 which is within experimental error of the value obtained at low per cent conversion, kH/kD = 1.156 -I- 0.026. The observed isotope effect at 100% conversion is unity. These data suggest that the intrinsic counting efficiencies of NMN and nicotinamide are equal under the counting conditions employed as has been demonstrated for other conditions by Cordes and co-workers' (26).

AMP nucleosidase is a very poor catalyst for NMN hydrolysis as evidenced by a turnover number of 2 s - l (5) . Therefore it must be established that enzyme-catalyzed hydrolysis, rather than base-catalyzed or uncatalyzed solvolysis, was observed in isotope effect determinations. Extrapolation of the data of Anderson and Anderson (52) and that of Cordes and co-workers (26) for the solvolysis of NAD to the conditions used in the isotope effect determinations assuming that the base-catalyzed pathway is the only kinetically important mechanism above pH 6.0 suggests that less than 1% of NAD is hydrolyzed in more than 3 h under these conditions. Incu- bation of NMN under conditions identical with those used in isotope effect determinations at pH 9.25 in the absence of enzyme resulted in a 5% yield of products in 1 h. This implies that in 4 h less than 1% ,of NMN is hydrolyzed at pH 8.00 under similar reaction conditions. Incubation of NMN in a reaction mixture at pH 8.00 in the absence of enzyme for 3 h yielded insufficient nicotinamide to be detected by ultraviolet absorption under conditions where a 0.3% yield would have been detected. In a similar experiment, no radioisotopically labeled nicotinamide was released from NMN after 4 h of

H. G. Bull, unpublished results.

incubation. The kinetic a-deuterium isotope effect on the uncatalyzed solvolysis of NMN is kH/kD = 1.135 f 0.019 (26). The isotope effects on the base-catalyzed hydrolysis of NAD and nicotinamide riboside are kH/kD = 1.151 f 0.012 and kH/ kD = 1.12 +. 0.01, respectively (27). Thus, a significant contribution to the observed isotope effects on the AMP nucleosidase-catalyzed hydrolysis of NMN by these nonenzymatic pathways should result in a large observed isotope effect. An isotope effect determination in which 90 min of incubation at pH 8.00 in the presence of 0.487 mM MgATP2- resulted in 0.30% yield of products and an isotope effect of kH/kD = 1.057 f 0.009. This isotope effect is within experimental error of values derived from other determinations under the same conditions but which resulted in greater product yield. These data establish that the contribution of nonenzymatic pathways to observed isotope effects for NMN hydrolysis in the presence of AMP nucleosidase determined at and below pH 8.00 is negligible.

NMN glycohydrolase activity has been observed in crude preparations of A. vinelandii (53, 54). Although one of these preparations contains AMP nucleosidase activity (53), the NMN glycohydrolase activity is activated by and dependent upon the presence of GTP or other guanosine nucleotides (53, 54), suggesting that the NMN glycohydrolase activity is independent of AMP nucleosidase. Incubation of [I4C]NMN in the presence of AMP nucleosidase and 1 mM GTP at pH 8.00, 30 "C, 2 mM MgClz resulted in the production of no more [14C]nicotinamide than did incubation of NMN in an enzyme- free reaction mixture. Incubation with AMP nucleosidase and 1.0 mM MgATP2- under identical conditions results in the recovery of [14C]nicotinamide. These observations suggest that the isotope effect determinations were not compromised by the presence of GTP-activated NMN glycohydrolase activity. At higher concentrations of enzyme and GTP, AMP nucleosidase-catalyzed hydrolysis of AMP and NMN can be activated by MgGTP2-,2 suggesting that the NMN glycohydrolase activity is due to AMP nucleosidase.

The observed isotope effect on the AMP nucleosidase- catalyzed hydrolysis of NMN in the presence of 1 mM Mg- ATP2- at pH 9.25 is a mixture of the isotope effect on the base- and enzyme-catalyzed pathways. The observed isotope effect may be corrected for the contribution of the base- catalyzed pathway by assuming that 5% yield/h is due to base catalysis, as indicated above, which by analogy to NAD and nicotinamide riboside hydrolysis is characterized by an isotope effect between 1.12 and 1.16 (27). These assumptions led to an isotope effect on the AMP nucleosidase-catalyzed hydrolysis of NMN at pH 9.25,l mM MgATP2- of kH/kD = 1.15 -I- 0.02.

Competitive determinations of isotope effects by the double label technique may be compromised by the depletion of a radioactive label from any species, labilization of deuterium, or scrambling of radioisotope-labeled bases among deuterated and unlabeled carbohydrate moieites. The isotopic composition of reactant samples before reaction and of reactant recovered from the reaction mixture was monitored in each determination. In no case do these compositions differ by more than 2%. This suggests that none of the radioactive labels are solvent labile. Lability of a label in either product or reactant but not both or enzyme-catalyzed release of radioactive labels would result in isotope effects which are dependent on the duration of incubation or the per cent yield, respectively. This is not the case for these isotope effect determinations. The result of synthesis of nucleotides from

' V. L. Schramm and F . A. Fullin, unpublished results.

Activation of AMP Nucleosidase-catalyzed Hydrolysis of AMP and NMN 4457

the pool of labeled bases and sugars in a reaction mixture or the labilization of deuterium in the reactant would result in the measurement of isotope effects approaching unity as the recovered reactant is isolated and reused repeatedly. The substrates used were recycled more than 20 times without noticeable diminution of the observed isotope effect. Catalysis of the synthesis of nucleotides from base and ribose 5-phosphate by AMP nucleosidase has not been observed under the conditions employed for these experiments (1, 7).

The errors reported for the isotope effects are derived from the propagation of the error in the determination of the number of counts accumulated for each sample through the equation used to calculate the observed isotope effect. This error is nearly always greater than the standard deviation of the isotope effect calculated from different countings and determinations. When the standard deviation is greater than the error determined from the counts accumulated, the standard deviation is reported as the error.

DISCUSSION

There are two general observations made in this work that must be accounted for: (i) the kinetic a-deuterium isotope effect on V,,,,,/K, for AMP nucleosidase-catalyzed hydrolysis of NMN at saturing concentrations of MgATP2- is near 1.15; the corresponding value for hydrolysis of AMP under the same conditions is near unity; and (ii) isotope effects for hydrolysis of NMN vary as a function of the degree of saturation of enzyme with MgATP2-; specifically, the isotope effect for NMN hydrolysis increases from near unity to about 1.15 with increasing concentrations of MgATP2-. That for AMP hydrolysis may decrease from about 1.04 to unity under these conditions, although it is not clear that this trend is real.

The large kinetic isotope effect for the hydrolysis of NMN catalyzed by fully activated AMP nucleosidase suggests that this reaction proceeds with partial or fully rate-determining cleavage of the N-glycosidic bond to yield nicotinamide and an oxocarbonium ion-like species derived from ribose 5-phosphate or with rate-determining reaction of this species. How- ever, oxocarbonium ions derived from glycosides are probably too short-lived in water to be intermediates in any reaction (55, 56). Therefore, participation of a nucleophile in the transition state is enforced (57, 58) and has been observed (57). Sinnott and Jencks (59) have observed nucleophilic participation in the transition state for solvolysis of glucopy- ranosyl glycosides in mixtures of ethanol and trifluoroethanol. In each case, nucleophilic participation is weak (57,59). This results in relatively unrestricted vibrations of the a-hydrogen in the transition state and observation of kinetic a-deuterium isotope effects between 1.05 and 1.18 in these reactions (57, 60). Therefore, the isotope effect for the hydrolysis of NMN is consistent with nucleophilic stabilization of the incipient oxocarbonium ion in the transition state. Future references to oxocarbonium ion character will include implicitly the possibility of nucleophilic participation.

The simplest mechanism for NMN hydrolysis, direct decomposition of the substrate through a transition state having oxocarbonium ion character, is related to that for structurally related substrates, including simple acetals (61, 62), O-glycosides (32, 33, 61-65), and purine and pyrimidine nucleosides (61, 66, 67). Nicotinamide nucleosides are cationic; the other species just indicated are generally uncharged in the ground state and must be protonated prior to covalent bond cleavage, reflected in acid catalysis for these reactions. Nucleophilic participation in the hydrolysis reactions of pyridinium nucleosides is reflected in the observation of base catalysis for

several nicotinamide nucleosides (52) and for 3-chloropyri- dinium-P-D-galactosides (68). As expected, the rate of hydrolysis of pyridinium glycosides increases with increasing electron withdrawal by substituents in the aglycone (26, 27, 68).

The kinetic isotope effect for NMN hydrolysis catalyzed by fully activated AMP nucleosidase is quite similar to those measured earlier for the uncatalyzed hydrolysis of several pyridinium nucleosides: B-galactopyranosyl pyridinium ion, k H / k D = 1.16 -t 0.04 (68), NAD, 1.101 f 0.005, NMN, 1.135 A 0.019 (26), and nicotinamide riboside, 1.144 f 0.008 (27). Since the uncatalyzed reactions necessarily exhibit the full intrinsic isotope effect for the isotope-sensitive step, this suggests that the value for the enzymatic reaction may also be the intrinsic one. That both uncatalyzed and enzyme- catalyzed reactions may involve nucleophilic participation is suggested by the observation that the base-catalyzed hydrolysis of NAD and nicotinamide riboside exhibits similar kinetic isotope effects: 1.15 and 1.12, respectively (27). The isotope effect for the enzymatic reaction is not changed detectably when water is replaced by deuterium oxide as solvent, an observation consistent with manifestation of the full intrinsic isotope effect for the isotope-sensitive step. Thus, it seems quite likely that the AMP nucleosidase-catalyzed hydrolysis of NMN occurs through an oxocarbonium ion-like interme- diate and that the formation or decomposition of this inter- mediate is largely ox fully rate-determining when the enzyme is fully saturated with MgATP2-.

Under the same conditions, the observed kinetic isotope effect for AMP hydrolysis is unity. Differences in isotope effects for different substrates might reflect one of two general possibilities: (i) variation in the relative rates of different steps in the enzymatic reaction pathway resulting in variable extents to which the intrinsic isotope effect associated with the isotope-sensitive step is revealed, or (ii) differences in transition state structure. The maximum isotope effect on AMP hydrolysis, k H / k D = 1.045, is consistent with an s,2 mechanism for this substrate. However, for the reasons detailed below, the former alternative of a change in rate- determining step seems more likely than different mechanisms for the two substrates.

There is evidence from both enzymatic and nonenzymatic studies that purine nucleosides hydrolyze via a mechanism similar to that already described for NMN: a-kinetic isotope effects for acid-catalyzed hydrolysis of adenosine, k H / k D = 1.23, and inosine, k H / k D = 1.20 (38), are large and near the calculated value for the equilibrium a-deuterium isotope effect for interconversion of protonated nucleosides and the corresponding oxocarbonium ions, k H / k D = 1.23 (39). Moreover, isotope effects for the phosphorolysis of adenosine and inosine catalyzed by purine nucleoside phosphorylases (20,21) suggest that these reactions, too, occur through the formation of an oxocarbonium ion-like species. Finally, it simply seems quite unlikely that the hydrolysis of NMN and AMP catalyzed by the same enzyme would proceed by radically different types of transition state. Thus, the different isotope effects seen for the hydrolysis of NMN and AMP very likely reflect varying contributions of the isotope-sensitive step to the overall rate of the reaction.

There is ample precedence for this situation with substrates very similar to those employed here. Specifically for NAD glycohydrolase from pig brain3 (27), Neurospora crassa (26), and bull semen: the kinetic a-deuterium isotope effect for NAD hydrolysis is unity; that is, the bond cleavage reaction is not rate-determining. In contrast, the isotope effects for

M. Sankovic, unpublished results. D. W. Visscher, unpublished results.

4458 Activation of AMP Nucleosidase-catalyzed Hydrolysis of AMP and NMN

the pig brain and N. crassa NAD glycohydrolase-catalyzed hydrolysis of NMN, kH/& = 1.132 f 0.010 and 1.100 rf: 0.008, respectively (26), are indicative of rate-determining bond cleavage and considerable oxocarbonium ion character in the transition states for these reactions. The isotope effect for the NMN glycohydrolase-catalyzed hydrolysis of NMN is unity (26), consistent with the view that glycohydrolases hydrolyze their natural substrates with a rate-determining step other than covalent bond cleavage. By analogy, this suggests that an isotope-dependent step is largely rate-determining for the hydrolysis of NMN catalyzed by fully activated AMP nucleosidase but contributes little or nothing to the determination of the overall rate of the same reaction for AMP.

The full intrinsic isotope effect for AMP nucleosidase- catalyzed hydrolysis of AMP is never observed. When the enzyme is less than half-saturated with MgATP2-, the isotope effect is apparently greater than unity, kH/kD = 1.043 f 0.010, suggesting that bond cleavage is partially rate-determining. As the enzyme becomes more fully saturated with MgATP2-, an isotope-independent step becomes wholly rate-determining and the observed isotope effect becomes unity when the concentration of MgATP2- is 1 mM. A decrease in isotope effect that starts at half-saturation is consistent with the existence of two classes of activator sites (9). Secondary tritium isotope effects for AMP hydrolysis catalyzed by AMP nucleosidases from A. uinelandii and E. coli show a similar trend, decreasing from 1.07 to 1.05 and from 1.09 to 1.05, respectively, as the concentration of MgATP2- increases to near-saturation at 0.5 mM (45).

The binding of substrates to AMP nucleosidase is not strongly dependent on the concentration of MgATP2- (4,69), suggesting that MgATP2- activates the enzyme principally by increasing the rate of conversion of ES to products rather than by stabilizing ES. Increasing the rate of conversion of ES to products relative to the rate of its dissociation to substrate and free enzyme will cause the isotope effect to approach unity (19), in accord with experimental observation.

The dependence of the isotope effect for NMN hydrolysis on the concentration of MgATP2- is also consistent with an activator-mediated change in the rate-determining step. When AMP nucleosidase is less than half-saturated with MgATP2-, kH/kD = 1.017 rf: 0.014, suggesting that an isotope- insensitive step is rate-determining. As the enzyme becomes more fully saturated with actyvator, this isotope-insensitive step is accelerated until an isotope-sensitive step is largely rate-determining. This model requires that the rate of the bond-changing step for NMN hydrolysis be less sensitive to the concentration of MgATP2- than the corresponding step in hydrolysis of AMP.

That NMN (26,52) is approximately 100-fold more reactive to nonenzymatic hydrolysis at pH 8 than is adenosine (70) suggests that NMN will be more reactive than AMP in the active site of AMP nucleosidase. Therefore, activation may have less effect on the rate of the bond-changing reaction for hydrolysis of NMN than for AMP hydrolysis. An interaction with N-7 of AMP (5) analogous to the protonation of N-7 of adenosine in acid-catalyzed hydrolysis (66, 67), which with- draws electron density from the reaction center, has been proposed to be essential to catalysis by AMP nucleosidase (5). However, the nitrogen of the N-glycosidic bond of NMN is positively charged, suggesting that this interaction is not essential to the AMP nucleosidase-catalyzed hydrolysis of NMN. It has been suggested that an interaction between enzyme and the amino group of AMP anchors the adenine moiety in the syn conformation about the N-glycosidic bond, resulting in destabilization of this bond (5). Comparison of

molecular models shows that the amide group of nicotinamide may occupy the same position as the amino group of AMP. However, there is no significant barrier to rotation of the nicotinamide moiety of NMN (71) or of the extended conformation of NAD (71-73) about the N-glycosidic bond. There- fore, interaction with the amide group of NMN may aid the reaction only through a small inductive effect. To the extent that activation of AMP hydrolysis depends on facilitation of the interactions outlined above, the rate of the chemical step for AMP nucleosidase-catalyzed hydrolysis of NMN will be independent of activation of the enzyme. The importance of these interactions is underscored by 2’-dAMP which has VI K only 1:40 that of AMP and exhibits a secondary tritium isotope effect much smaller than the intrinsic isotope effect, k H / k T = 1.08 (45). This suggests that although the 2”hydroxyl group of AMP is required for optimal enzyme activity, the features of the adenine moiety that NMN lacks are essential to enzymatic acceleration of the bond-cleavage step. That AMP nucleosidase catalyzes the hydrolysis of NMN suggests that factors other than these are partially responsible for catalysis.

The isotope effect on AMP nucleosidase-catalyzed NMN hydrolysis, like the isotope effect on AMP hydrolysis (45), is independent of pH which indicates that the pH-dependent and isotope-sensitive steps of the enzyme mechanism are identical (24, 25). The dependence of kinetic constants for AMP hydrolysis and the inhibition constant for FMP on pH have implicated the 5‘-phosphate, a group on the enzyme of pK. approximately 6.6, and two groups on the enzyme of pK, near 8.2 in catalysis by AMP nucleosidase (5). These groups may be those responsible for the essential interactions with N-7 and the amino group of AMP. The incipient oxocarbonium ion would be stabilized by interaction with a carboxylate ion in the active site whose pK, has been perturbed to 6.6. While the pH dependence of kinetic constants for AMP nucleosidase has been established (5), the pH-independent isotope effects indicate that one of the titratable groups is involved in the bond-breaking step.

Acknowledgments-I am indebted to the following scientists: Dr. E. H. Cordes for the guidance and support through this work that lead to my degree; Dr. M. Sankovic for assistance in the synthesis of [c~rbonyl-’~C,l’-~H]NAD; at Temple University: Dr. V. L. Schramm for unpublished data, advice, and fruitful discussions; Dr. D. Parkin for determination of the 3H isotope effect for AMP nucleosidase- catalyzed hydrolysis of AMP in the absence of MgATP2-, and F. A. Fullin for determining the initial rates of NMN hydrolysis.

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mechanism and activation for allosteric adenosine 5 ...at secondary centers usually exhibits isotope...

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