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Indian Journal of Biochemistry & Biophysics Vol. 38, February & April 200 I, pp. 34-4 1
Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution
Sujata Sharma' , Renu Tyagi 2, M N Gupta2 and T P Singh' -
'Department of Biophysics, All India Institute of Medical Sciences, New Delhi 110 029, India
2Department of Chemistry, Indian Institute of Technology, New Delhi 11001 6, India
Accepted 3 October 2000
For the first time, it is demonstrated that exposure of an enzy me to anhydrous orga ni c solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat ge rm acid phosphatase, a-alllylase, l3-g lucosidase, chymotrypsin and trypsi n were exposed to aeetonitrile at 70°C for three hr. The ac tivities of these enzymes were found to be considerably enhanced. In order to understand the basis of thi s change in the acti vity of these enzymes, proteinase K was analyzed in detail us ing X-ray diffraction method. The overall structure of the enzyme was found to be si milar to the nati ve structure in aqueous environment. The hydrogen bonding system of the catalytie tri ad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrange ment as some of the water molecules were ei ther di splaced or completeiy absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. In terlinked th rough water molecules, the sites occupied by acetonitrile molecu les were independent of water molecules . The acetonitrile molecules are in volved in ex tensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN I) interacts si multaneously with the hydrophobic side chains of Leu 96, lie 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in lie 107 by rotat.ing its side chain about Ca- CI3 bond by J 80° to bring about the o-methyl group within the range of attractive van der Waals interac tions with the methyl group of CCN I. A similar change had earlier been observed in proteinase K when it was complexed to a substrate analogue, lac toferrin fragment.
Introduction The analysis of enzyme behaviour in anhydrous
media has important implications in biotechnology and organic chemistryl -4 . Some interesting phenomena like pH memor/·6 and molecular imprinting7•8 utilizing such media have also been reported. The key issue is to evaluate the effects of exposure to such media on the structure of the enzymes. There have been reports on enzyme structures by soaking the crystals of enzymes which were grown in aqueous media, in organic solvents using methods of X-ray diffraction9- 11 , NMR 12 and FfIR 13. The results of these investigations did not indicate any significant structural change in the protein molecule and both flipping rates and liberational motions in the enzyme were found much greater in aqueous crystals rather than in lyophilized
*Author for correspondence E-mail : tps @aiims.aiims.ac.in
powders suspended in anhydrous solvents 12. It may be recalled that the enzymes when placed in such media showed exceptionally high thermal stability which is monitored by their biological activit/ 4• With the prevailing notion that nothing drastic seems to happen to the enzyme structures in organic solvents, we conducted experiments by placing the enzymes in anhydrous organic solvents at a rnoderately high temperature of around 70°C. We report here the changes in the kjnetic parameters of six different enzymes as a result of such an exposure and three- dimensional (3 D) structural changes in one of them viz proteinase K, a serine proteinase from fungus Trirachium album Limber l5 .
Materials and Methods Trypsin, wheat germ acid phosphatase, a-amylase,
~-glucosidase, p-nitrophenyl, ~-·glucopyranoside (PNGP), N-a-benzoyl DL-arginine p-nitroanilide (BAPNA) and N-a-benzoyl arg in ine ethyl ester
SHARMA el 01.: 3D STRUCTURE OF MODIFIED SERINE PROTEINASE 35
(BAEE) were purchased from Sigma Chemical Co. USA. Chymotrypsin and p-nitrophenyl phosphate (PNPP) were purchased from Sisco Research Lab., India. Proteinase K was obtained from E.Merck, Germany. Organic solvents were of HPLC grade and llsed after drying over 4 A molecular sieves. All other reagents used were of analytical grade.
Heat treatment of enzymes in organic solvent The enzyme (l mg) was put in different 10 ml
screw capped vials. One ml organic solvent (aceto- nitrile/toluene/dimethyl fonnamide) was added to each vial and the enzyme solvent suspension was heated at 70°C for different time intervals (0-5 hr) in a water bath. The samples were cooled at 25°C and the solvent was removed using vacuum (Tables 1 and 2).
Enzyme assays Activities of proteinase K 16 and chymotrypsin 17
were measured using BTEE as substrate. Activities of acid phosphatasel 8, a-amylase 17, ~-glucosidaseI9 and trypsin20 were measured using PNPP, starch, PNGP and BAPNA as substrates respectively. All the activity measurements were repeated six times. The values showed a variation of less than 3%.
Table I-Effect of heat treatment on enzymes in acetonitrile
[The enzyme (1 mg) was incubated with acetonitrile (1 ml) at 70°C for varying time intervals (0-5 hr) and activities were measured after the removal of solvent as described. Control was also run where the organic solvent was removed after 30 seconds of incubation at 25°C. The initial rates were calculated with those samples where maximum increase in the activity was observed]
Enzymes Time V/ Vc# VTIVC (hr)*
Proteinase K 3 48 35 1.37
Chymotrypsin 2 60 48 1.25
Acid phosphatase 5 40 36 1.11
~-glucosidase 4 260 164 1.59
Trypsin 3 67 53 1.32
a-Amylase 3 42 36 1.16
*Time of incubation for maximum enzyme activity. #VT and Vc are the initial rates (in Jlmole min·
t mg' t protein) of the product formation for treated and control samples respectively.
Crystal preparation Proteinase K (E.e. 18.104.22.168) from fungus
Tritirachium album Limbert5 obtained from Merck (Darmstadt, Germany) and treated with organic solvents as described earlier was purified by gel filtration using Sephadex G-75 column in 50 mM Tris.HCl, pH 7.5 containing 1 mM CaCI2. Fractions of highest activity were pooled, dialyzed exhaustively at 4°C against 1 mM calcium acetate and lyophilized. This treated and purified form of proteinase K was crystallized by dissolving the 10% (w/v) of lyophilized enzyme in 50 mM Tris.HCl, 1 mM CaCI2, H 6.5. This solution (25 III drops) were equilibrated in a microdialysis set-up against 1 M NaN03 in the same buffer at 4°e. The crystallization set-up did not contain organic solvents. Single crystals of size 0.3xO.2xO.2 mm3 grew within 6-7 days.
X-ray diffraction data For X-ray intensity data collection, one crystal was
mounted in a glass capillary. The X-ray intensities were collected to 2.2 A resolution at 12°C using a 300 mm MAR Research imaging plate scanner mounted on a RU-200 Rigaku rotating anode generator operating at a rate of 40 kY and 100 mAo The graphite monochromater was used to generate the CuKa radiation. Crystallographic data, data collection and processing details are given in Table 3. The data were processed using the HKL-package2t .
Results and Discussion
Analysis of kinetic data It has been shown that the enzymes in anhydrous
organic solvents show considerably higher themo- stability as compared to their stability in aqueous buffers 14. It has also been reported that at considerably high temperatures, viz. 11O-145°C, the enzymes such as ribonuclease, chymotrypsin and lysozyme lose their activity by following inactivation mechanisms similar to the ones established for thermo-inactivation in aqueous buffers22 . However, no data were available about the effect of heating on the enzymes in such media at temperatures which are moderately high but still not high enough to cause significant loss of enzyme activity.
Six enzymes, viz. Proteinase K, chymotrypsin, acid phosphatase, ~-glucosidase, trypsin and a-amylase were heated at 70°C in nearly anhydrous acetonitrile for a few hours. After this treatment, the enzymes were cooled to room temperature and organic solvent
36 INDIAN J BIOCHEM BIOPHYS, VOL. 38, FEBRUARY & APRIL 2001
was removed by placing the system under reduced pressure. The initial rates of respective substrate conversions are shown in Table 1. In all the cases, the "exposed" enzymes showed higher in itial rates of activities as compared to the "controls". These observations are in agreement with an earlier preliminary report on urease, N,N-dimethyl
formamidase and phospholipase A/3. So far, chemical modifications24 and protein engineering25
had been used to alter the catalytic power of the enzymes. The semjnal work with triose phosphate isomerase26 has emphasized that the evolutionary forces tend to optimize kca/Km in thei r search for the perfect enzyme.
Table 2- Effect of heat treatment in organic solvents on VmaJKm of enzymes