THE JOURNAL OF n l O L o G l C a CHEMISTRY

Printed u1 U.S.A. Vol. 257, No. 11, Issue of June 10, p p . 6420-6126. 1982

Comparison of Amino Acid Sequence and Thermostability of Tyrosinase from Three Wild Type Strains of Neurospora crassa*

(Received for publication, November 4, 1981)

Christoph Ruegg, Doris Ammer, and Konrad Lerch$ From the Biochemisches Znstitut der Uniuersitat Zurich, Zurichbergstrasse 4, CH-8028 Zurich, Switzerland

The thermostability of tyrosinase from three wild type strains of Neurospora c m s a has been investi- gated. For this purpose a sequence comparison of two thermostable and one thermolabile tyrosinase isoen- zyme was carried out. It revealed that at position 201 the thermostable enzyme forms share an aspartate res- idue in contrast to an asparagine residue in the ther- molabile form. In addition, one of the thermostable isoenzymes displays five other substitutions. Since the relative stability of the thermostable forms as com- pared to the thermolabile one decreases with increas- ing ionic strength, the common aspartate residue is thought to bring about the additional stability of the thermostable isoenzymes by forming a salt bridge be- tween aspartate 201 and a positively charged group of the protein. The strong pH-dependency of the thermo- stability with an apparent pKa of 6.6 indicates a histi- dinium side chain as the most likely ionic group to be involved in the salt bridge. This conjecture is also sup- ported by measurements of the stability towards the chaotropic agent guanidinium chloride. The difference of the free energy change of denaturation AGDHz0 be- tween the apoenzymes of a thermostable and a ther- molabile isoenzyme was calculated as 2.5 kcal mol“. Furthermore, it was shown that the copper ions of the native and the cobalt ions of Co(I1)-substituted tyrosin- ase strongly enhance the stability of the protein as compared to its apoform.

Proteins are generally stable only within a limited temper- ature range (1). Above a certain temperature (around 50 “C) most enzymes are inactivated quickly. Certain organisms, however, live at an elevated temperature and they synthesize proteins which are stable even at their high ambient temper- atures (60-95 “C) (3, 4). It was also found that some proteins of mesophilic organisms show an increased thermostability (5, 6). This was correlated inversely to the size of the protein molecules. The higher thermostability was also related to increasing accessible surface area of the protein molecule (6). To explain the extra stability of the proteins from thermo- philic organisms, different mechanisms have been proposed. Perutz (2) stressed the influence of extra salt bridges on thermostability in a recent article dealing with electrostatic interactions in proteins. Argos et al. (7) showed an increased internal hydrophobicity in the proteins from thermophilic organisms. An increased number of hydrogen bonds has been

* This work was supported by Schweizerischer Nationalfonds Grant 3.420-0.78. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

k, To whom correspondence should be addressed.

proposed by Barnes and Stellwagen (8) to account for ther- mostability. Common to all mechanisms is that small changes in protein structure suffice to bring about a remarkable in- crease of thermostability. Neurospora tyrosinase, a copper- containing monooxygenase of molecular weight 46,000 (9), is important for the formation of melanin pigments and is syn- thesized during sexual differentiation. The enzyme has been isolated from different wild-type strains of Neurospora crassa. These forms do not differ in their catalytic properties but they display remarkable differences in their thermostability and electrophoretic mobility (10, 11). Already in 1953 Horowitz and Fling (12) had shown by genetic analysis that the different isoenzymes are allelic forms. The thermolabile enzyme was reported to have a half-time of inactivation of 4 min at 60 “C (pH 6.0) whereas the thermostable forms TS and Sing have an inactivation half-time of 70 min (10).

Since the catalytic properties are identical, the differences in structure probably stem from parts of the protein molecule other than the active site. The primary structure of the enzyme from the thermolabile strain TL has been reported (9, 13,14). In this study, a sequence comparison together with some physicochemical measurements of the thermostability of tyrosinase from the three Neurospora wild type strains TL, TS, and Sing was used to investigate some aspects of ther- mostability. The results lead us to propose that Asp 201 forms a salt bridge to a histidine residue in the thermostable enzyme forms. This salt bridge is thought to account for their addi- tional stability.

MATERIALS AND METHODS‘

RESULTS

Sequence Comparison-In order to accomplish a compari- son of the amino acid sequences of tyrosinase from the strains TS and Sing to the known sequence of strain TL, the following strategy was applied. The enzymes were first cleaved with cyanogen bromide and the resulting fragments separated by gel filtration on Sephadex G-100 and G-50 (13, 14). The NH2- terminal sequences of the intact cyanogen bromide fragments (except CB3 which is blocked) were established by automated sequence analysis. With the exception of CB4, all fragments were further subjected to tryptic digestion and the peptides separated by a combination of ion exchange chromatography, gel filtration, high voltage electrophoresis and thin layer chro- matography. The cyanogen bromide fragment CB1 was also

Portions of this paper (including “Materials and Methods,” and Tables VI-XXI) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MI> 20814. Request Doc- ument No. 81-2703, cite authors, and include a check for $7.60 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

6420

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Tyrosinase Isoenzymes: Primary Structure and Thermostability 6421

FIG. 1. The strategy for the se- quence comparison is outlined in this scheme. Tryptic (T), peptic ( P ) , and therrnolyt,ic (Th) peptides were gen- erated from the cyanogen bromide frag- ments CB1 to CB4 as indicated. The stippled areas of the bars represent the parts of the peptides which were sub- jected to Edman degradations.

TABLE I Amino acid composition of the peptides differing in the three isozymes of Neurospora tyrosinase

Peptide

Amino acid CB3-T6 from CB1-T1 from CB1-TI0 from CB2-T6 from

TS TL" Sing TS T L Sing TS T L Sine TS TL." Sine

Aspartic acid 3.0 Threonine (0) 1.Oh 0.9 (1) 1.0 2.0 (2) z h

Serine (4) 4.9O

3.1 (3) 3.1 1.0 (1) 1.1 Glutamic acid 4.2 1.1 (1) 1.0 2.3 (2) 2.1 Proline 1.1 (1) 1.1 1.1 (1) 1.0 2.6 (3) 3.0 0.9 (1) 1.1

(I) 1.1 Glycine 1.4 2.0 (2) 2.1 1.2 (1) 1.1 1.2 (1) 1.0 Alanine 1.9 (2) 2.0 1 .o (I) 1.0 Valine 1 .0 (1) 1.1 (1) 1.0 Isoleucine

(5) 5.0 0.9

Leucine 5.1 1.0 (1) 1.0 (1)

Tyrosine 1.8 (2) 2.0 Phenylalanine 1.0 (1) 1.0 0.8 (1) 1.1

1.8 (2) 1.6

Histidine 3.0 (3) 3.1 1.1 (1) 1.0 1.0 (1) 1.0

Lysine Arginine 1.0 (1) 1.0 1.0 (1) 1.0 1.0 (1) 1.0

~ ~~ ~ ~~~~

(3) 2.0" 2.4 (2) 2.1 1 .o (1) 1.1 -

-

1.0 (1) 0.9

V: 0.069 0.068 0.060 0.420 0.420 0.423 0.425" 0.458 0.435' 0.126 0.129 0.125 Residues 29-46 127-134 - 190-208 - 368-381

values are shown in parentheses. The amino acid composition of the peptides from strain TL is derived from the sequence determined by Lerch et al, (13, 14). Integral

Numbers differing from those of strain TL are underscored. " v, is defined as the ratio between the elution volume of the peptide and the total volume of the gradient,

digested with pepsin and fragment CB4 with thermolysin. The strategy is outlined in the scheme presented in Fig. 1.

In Table I, those peptides of the thermostable strains TS and Sing are listed which differ either in elution volume from the cation exchange resin ("72) or in amino acid composition from the corresponding peptide of TL tyrosinase. From man- ual Edman degradation of the tryptic peptide Sing CB1-T1 (residues 127-134) the third amino acid was found to be a threonine residue instead of valine 130 in the corresponding TL peptide. A threonine residue was again identified in pep- tide Sing CB2-T6 (residues 368-381) at the third position which replaces isoleucine 370 of the corresponding TL peptide.

Automated Edman degradations of peptide Sing CB3-T6 (residues 29-46) showed aspartate residue 29 of the corre- sponding TL peptide to be replaced by a glutamate residue. Two more substitutions were found in the NHp-terminal part of Sing CB3 (residues 326-407), where the adjacent residues Lys 345 and Ser 346 of TL and TS tyrosinase are replaced by a glutamine and an asparagine residue, respectively. In both peptides TS CB1-T10 (residues 190-208) and Sing CBl-TlO (residues 190-208) an aspartate residue was identified at the twelfth position in contrast to an asparagine residue of the corresponding TL peptide.

The amino acid composition and elution volume from the cation exchange resin "72 of all the isolated peptides are presented in the miniprint section (Tables VI-X) together with the results of the different manual and automated Edman degradations (Tables XI-XXI). The complete amino acid sequence of Neurospora tyrosinase TL is shown in Fig. 2 with the substitutions for the allelic forms TS and Sing indicated by a circle and by boxes, respectively. The sequence differ- ences of the three allelic forms are summarized in Table 11.

Investigation of Stability Differences-Tyrosinase of the three strains TL, TS, and Sing display the same specific activity and the same Arrhenius activation energy of the enzyme-catalyzed reaction (Table 111). Furthermore, the three isoenzymes reacted positively in the Ouchterlony test towards antibodies directed against TL-tyrosinase in agreement with earlier experiments of Katan et al. (15). However, the three forms differ markedly in the Arrhenius activation energy of the heat inactivation, their isoelectric point (Fig. 3 ) and their stability towards chaotropic agents. These differences were further investigated.

Effect of Phosphate Concentration on the Relative Stabil- ities-The relative stabilities, i.e. the ratios of the half-times of the heat inactivation of the pairs Sing/TL and TS/TL at

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6422 Tyrosinase Isoenzymes: Primary Structure and Thermostability

C S L ~ A V t i N E l t i D R l G G N G Y w S S L E V S A F O ?

L F H L ~ H V Y V D R L W S I Y Q D L N P ~ ~ S F ~ I ~ R P A

P Y S l ~ V A Q ~ ~ E S ~ S ~ ! P l E D F w c a s n n N F

~ l S E O V L D S ~ 1 F G Y A Y P E I Q Y Y w Y S S ' ~ ~ ~ Y

O A A I R K S V I ~ ~ L Y G S N V F - c ; J

FIG. 2. Primary structure of Neuroslom tyrosinase from strain TL (9). The substitutions for the thermostable isoenzymes are indicated by a circle for strain TS and by boxes for strain Sing. For the one-letter notation for amino acid residues, see J. Biol. Chem. 243. 3557-3559 (1968).

TABLE I1 Amino acid residues differing in the three allelic forms of

Neurospora tvrosinase

Enzyme - Position of substitution

29 129 20 1 34G 370 345 form ____ ""

T L Asp Val Asn Lys Ser Ile TS Asp Val Asp Lys Ser Ile Sing Glu Thr Asp Gln Asn T h r

. ~ _ _ ~ _ _ _ _ _ _ _ _ _ _ " ~ _ _ _ _ ~

TABLE I11 Characterization of tyrosinase isoenzymes

form Specificactivity KM" 6 ,,.,, *," E., ,h, ' ( I 1.' IEP" "~ ~~~

Enzyme ~.

_ _ _ ~ ~ -~ ~~ . _ _ ~ ~ units mg m .w kc01 mol I

Sing 1290 f 150 1.09 12.1 98 60 8.0 1's 1340 f 140 0.88 11.9 107 60 8.3 TL 1330 f 100 0.95 12.0 80 4 8.5 '' Determined in 0.1 M sodium phosphate, pH 6.0, using I.-I)OPA as

" Arrhenius activation energy of the enzyme catalyzed reaction

' Arrhenius activation energy of heat inactivation. " At 60 "C in 0.1 M sodium phosphate, pH 6.0. " Isoelectric point.

substrate.

with I.-I)OI'A as substrate.

pH 6.0 are plotted against the logarithm of the ionic strength of the sodium phosphate buffer (Fig. 4). Below 0.1 M the ratio is around 16 and decreases monotonically to a value of ap- proximately 8 a t 1.1 M.

Effect ofpH on Thermostability-Fig. 5 shows the stability ratios of the thermostable enzyme forms compared to the thermolabile one as a function of pH a t constant ionic strength of I = 0.1.? Up to about pH 6.2 these ratios were found to be around 16. However, above pH 6.2 there is a sharp decrease of the relative stability from 16 to 2 at pH 8 with an apparent

Denaturation by Guanidinium Chloride-To compare the stability of the three isozymic forms against the chaotropic agent GdmC1. the enzymatic activity and the magnitude of the CD-band at 224 nm were followed at different concentra-

'The ahbreviations used are: I , ionic strength; GdmCI, guanidinium chloride; GLC, gas liquid chromatography: HI'LC, high pressure liquid chromatography; 1,-1>01'A, 1.-3,4-dihydroxyphen~Ialanine: I'TH, phenvlthiohydantoin; K, regeneration: TLC, thin layer chro- matography: tl!'; half-time, CH, cyanogen bromide.

PK,~ of 6.6.

TL v TS F Sing v

FIG. 3. Isoelectric focusing of the three tyrosinase isoen- zymes on an agarose gel. 5-1Opg of protein were applied.

001 01 6 1 0

loncc Slvenplh IMI DH

FIG. 4, left. Influence of ionic strength on the relative ther- mostability at 60 "C. Ratios of inactivation half-times of thermo- stable to thermolabile enzyme forms in phosphate buffer. pH 6.0. are plotted versus ionic strength. Enzyme activities were measured using I.-DOPA as substrate. 0, t i > ; r d I . 2 : I . I . : 0. t l ~ , s ~ , , ~ : / I 2 ; ~ ~ , .

FIG. 5, right. Dependence of the relative stabilities on pH in sodium phosphate buffer of I = 0.1 M at 60 "C. Ratios of inacti- vation half-times of thermostable to thermolabile enzyme forms in appropriate phosphate buffer are plotted c'ersus pH. Enzyme activi- ties were measured using I.-I)OI'A as substrate. 0. tI L:rh:tt LTI . : 0. tI ?.JIIIL:tl 2:l.l..

" I I

1 2 3 4 1 2 3 4 Concentration (MI C o n c e n t r a t i o n (MI

FIG. 6. The relative stabilities of the enzyme pair TS/TL (closed symbols) and Sing/TL (open symbols) are plotted against GdmCl concentration at pH 6.0 (circles) and pH 7.5 (triangles). A, inactivation at 0 "C is monitored by activity measure- ments. B, denaturation at 5 "C is measured by CD.

tions of GdmCl and at 0 and 5 "C, respectively. It was found that the decrease of these two parameters follows first-order kinetics and therefore the half-time of decrease t i was used as a measure of stability. At pH 6 the ratios of tl 2 of the thermostable forms to the thermolabile one are about i for the enzymatic inactivation and about 4.5 for the CD-band decrease. However, at pH 7.5 this ratio is diminished to about 3.5 for enzyme activity and 2.0 for the overall structure man- ifested by the ellipticity at 224 nm (Fig. 6).

The half-times of GdmCI-denaturation of TS tyrosinase as an example vary between 6000 min for 2 M GdmCl and 42 min for 4 M GdmCl at pH 6. To investigate the relationship between the decrease of enzymatic activity and the decrease of ellipticity at 224 nm, the half-times of denaturation were determined in 4 M GdmCl at pH 6.0 and at 5 and 10 "C (Table

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Tyrosinase Isoenzymes: Primary TABLE IV

Comparison of the rates of enzyme inactivation and decrease of ellipticity at 224 nm and at 5 and 10 "C in the presence of 4 M

GdmC1, p H 6.0 Enzyme

form T Method of measurement tl/? tl,n. a<t/t1,2. CI)

"C nin TS 10 CD 10.5 TS 10 Activity 9.5 TS 5 CD 16.1 TS 5 Activity 14.6 TL 10 CD 2.0 TL 10 Activity 1.7 TL 5 CD 2.5 TL 5 Activity 2.4

0.90

0.91

0.87

0.96

0 1 2 3 4 1 2 3 4

FIG. 7, left. The extent of denaturation measured by CD of apotyrosinase from strains TL (0) and TS (0) is plotted as a function of GdmCl in 0.1 M sodium phosphate, pH 6.0, at 5 "C.

FIG. 8, right. The free energy of denaturation AGD at 6 "C (calculated from Fig. 7) is plotted versus the GdmCl concentra- tion for strains TL (0) and TS (0).

Concentration (MI Concentrat ion ( M )

TABLE V Comparison of denaturation rates of different enzyme derivatives

in GdmCl measured by circular dichroism Enzvme derivative t , ,>"

Apotyrosinase Native tyrosinase Co(I1)-tyrosinase

min

220 70

1.6

4 "C " Measured in 0.1 M sodium phosphate, pH 6.0, + 3 M GdmCl at,

IV). I t was found that the enzymatic activity decreases about 10% faster than the ellipticity a t 224 nm.

Stability of Apotyrosinase-The denaturation curves of apotyrosinase from strains TS and TL (Fig. 7) obtained by CD-measurements a t 224 nm show again an increased stability of the thermostable form as compared to the thermolabile one. Since the unfolding process of apotyrosinase is much faster than that of holotyrosinase it was experimentally pos- sible to obtain the complete denaturation curves rather than the denaturation rates measured for the holoenzymes. The GdmCl concentration at which the protein is 50% unfolded was found to be 1.8 and 2.4 M for the apotyrosinase forms TL and TS, respectively.

The free energy change of denaturation, A G D ~ ~ O , was eval- uated from the denaturation curves using the linear extrapo- lation procedure described by Pace (16). These energies are 2.35 kcal mol-' for TL- and 4.9 kcal mol" for TS-tyrosinase (Fig. 8). O G U ~ ~ ~ was also calculated using the denaturant binding model of Aune and Tanford (17). The AGDH2' values

Structure and Thermostability 6423

obtained with this method were 2.7 and 5.2 kcal mol-' for the isoenzymes TL and TS, respectively.

Influence of Metal Ions on Protein Stability-A compari- son of the rates of unfolding between holo-, apo- and Co(I1)- substituted tyrosinase from strain TS was carried out to study the influence of the metal ions present at the active site. The half-times of denaturation as measured by the decrease of the ellipticity at 224 nm are presented in Table V.

DISCUSSION

Tyrosinases from the three wild type strains TS, TL, and Sing do not differ functionally. They all have the same KM for the substrate L-DOPA and the same Arrhenius activation energy of the enzyme-catalyzed reaction (Table 111). Further- more, the three isoenzymes also cross-react immunologically in the Ouchterlony test. They differ, however, in properties not directly related to their function. The three isoenzymes have different isoelectric points, different Arrhenius activation energies of heat inactivation, and different stabilities towards denaturation by heat or GdmC1. These differences must be reflected in the primary structure of the molecule. The se- quence comparison of the three allelic forms of Neurospora tyrosinase shows that the only difference between tyrosinase TL and TS lies in the substitution of Asn 201 of the thermo- labile form TL by Asp in the thermostable form TS. The differences of the physicochemical properties between the two isoenzymes must therefore be the result of this amino acid substitution. The other thermostable form, Sing, shares this substitution and contains in addition five more substitutions, i.e. Asp 29 --* Glu, Val 129 --* Thr, Lys 345 -+ Gln, Ser 346 + Asn, and Ile 370 --* Thr (Table 11).

The isoelectric focusing experiments (Fig. 3 ) showed lower isoelectric points for the thermostable enzyme forms, in agree- ment with the presence of an additional acidic amino acid residue. Furthermore, Sing tyrosinase lacks lysine residue 345 and consequently displays the lowest isoelectric point of the three isoenzymes.

From a comparison of the amino acid sequences from en- zymes of thermophilic to mesophilic organisms, Argos et al. (7) proposed that thermostability is often caused by an in- creased amount of hydrophobic amino acid residues in the interior of the protein molecule. Since the substitutions Val * Thr, Ile --* Thr, Asp "+ Glu, Lys += Gln, and Ser += Asn decrease rather than increase the hydrophobicity of the en- zyme form Sing, this mechanism of increased internal hydro- phobicity cannot account for the thermostability of the Sing isoenzyme. The common substitution Asn 201 "+ Asp found for the two thermostable isoenzymes is therefore thought to be responsible for the difference in stability.

An aspartate residue would offer the possibility to form an additional salt bridge to a nearby positive charge. Additional salt bridges have been proposed to give extra stability to enzymes in the order of about 1 to 3 kcal/mol of enzyme ( Z ) , which would easily account for a 10-fold increase of tIl2. If such a salt bridge is indeed responsible for the increased thermostability in the two forms TS and Sing it should be weakened by the addition of free ions. The measurements of the relative thermostability as a function of the ionic strength (Fig. 4) at pH 6.0 showed that the ratio of stability indeed decreases with increasing ionic strength.

TO get some insight into the possible counterion forming a salt bridge with aspartate residue 201, the relative thermost- abilities were measured as a function of pH. They were found to decrease with increasing pH (Fig. 5). The apparent pKa of 6.6 obtained from the ratios of thermostability (Fig. 5) suggests a histidine residue as the most likely counterion. Although it is known that pKA values of amino acid side chains in proteins

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6424 Tyrosinase Isoenzymes: Primary Structure and Thermostability can vary considerably, the large difference of four pH units between 6.6 and the pK,4 value of lysine makes this amino acid residue a very unlikely candidate as the counterion to Asp 201.

To rule out that the results above reflect an unspecific effect of phosphate ions, which often bind to proteins, the thermo- stability measurements were also carried out in the nonbind- ing buffer cacodylate. Virtually the same results were obtained (data not shown); i.e. the relative stability decreased with increasing ionic strength and showed the same pH dependency with an apparent pKa of 6.6.

Differences in stability between the three isoenzymes were not only manifested by thermal denaturation but also by denaturation with the chaotropic agent GdmC1. It was found that the thermolabile tyrosinase TL is unfolded more rapidly than the two thermostable tyrosinases TS and Sing. The finding that the relative stability is again pH-dependent fur- ther supports the idea that an additional salt bridge is respon- sible for the increased stability of the thermostable isoen- zymes. The denaturation in GdmCl was followed either by activity measurements or by measurements of the ellipticity at 224 nm. Since the activity decreases faster than the ellip- ticity at 224 nm, denaturation by GdmCl presumably is not a simple two-state process which involves only the native and the denatured state. Intermediates must be present which are enzymatically insctive but nevertheless show considerable secondary structure (16).

The large differences in stability towards the chaotropic agent GdmCl between apotyrosinase and the metal containing Co(I1)- or holotyrosinases suggest that the active-site metal ions play an important role in stabilizing the structure of the enzyme by linking ligands from quite different parts of the primary structure of the protein molecule. Stabilizing effects by metal ions have been observed in other metalloproteins, for instance in Cu/Zn containing superoxide dismutase (18) or thermolysin (19). From the denaturation curves obtained for apotyrosinase from strain TS and TL (Fig. 7) the Gibbs free energy change of denaturation AGD~'' was calculated using two different methods (16, 17). With both methods, a difference of AGDHa0 between the 2 isoenzymes of approxi- mately 2.5 kcal mol" was calculated. A salt bridge at the surface of a protein can contribute up to 1 kcal mol-' of stabilizing energy (2) whereas an internal salt bridge in chy- motrypsin added 3 kcal mol" (20). Therefore, the putative Asp 201-His x salt bridge of the thermostable strains is more likely to be located in the interior of the protein.

Polymorphism has been reported for numerous species of plants and animals (21) as well as in Escherichia co2i popu- lations (22, 23) and other bacteria (24). Tyrosinase from N . crassa represents an example of polymorphism in a eucaryotic microorganism. This polymorphism was reported already in 1961 by Horowitz et al. (10). In the present study it was investigated at the molecular level for three wild type strains of N. crassa. One of the variants (Sing) shows 5 and 6 substitutions as compared to TS and TL, respectively. This is a high number of substitutions between isoenzymes of the same species. Because all three tyrosinase isoenzymes possess the same catalytic properties, this enzyme seems to be tolerant towards a high degree of alteration without becoming func- tionally impaired. Five of the six substitutions observed (Asp 201 -f Asn, Ile 370 3 Thr, Asp 129 "-f Glu, Lys 345 -+ Gln,

and Ser 346 Asn) can be explained by single point mutations while the sixth substitution Val 129 "+ Thr involves at least two base changes.

The great variability of the primary structure of tyrosinase is further corroborated by preliminary sequence studies with another isoenzyme from strain PR, which displays still an- other substitution (Val 307 + Ile) in the fragment CB4."

Tyrosinase is an enzyme involved in the secondary metab- olism of many fungi. It is required for the synthesis of melanin pigments during sexual differentiation. Therefore it is quite dispensable. This would allow for fast sequence changes con- sistent with the view of Wilson et al. (25), who proposed that the evolutionary rate of a protein depends both on the amount of functional constraints and on the dispensibility of the protein.

REFERENCES 1. Nojima, H., lkai, A., Noda. H., Hon-Inami, K., and Oshima. T.

2. 3.

4.

5.

6. 7.

8.

9. 10.

11. 12. 13.

14. 15.

16. 17. 18.

19.

20.

21. 22. 23. 24.

25.

26. 27.

28. 29.

30.

(1978) in Biochemistry of Thermophily (Friedman, M. S., 'ed) pp. 305-323, Academic Press, New York

Perutz, M. F. (1978) Science 201, 1187-1191 Zuber, H. (ed) (19761 Enzymes and Proteins from Thermophilic

Microorganisms, Birkhauser Verlag, Base1 Brock, T. D. (1978) Thermophilic Microorganisms and Life at

Bull, H. B., and Breese, K. (1973) Arch. Biochem. Biophys. 158, High Temperatures, pp. 51-58,311, Springer-Verlag, New York

Stellwagen, E., and Wilgus, H. (1978) Nature 275, 342-343 Argos, P., Rossmann, M. G., Grau, U., Zuber, H., Frank, G., and

Barnes, L. D., and Stellwagen, E. (1973)Biochemistry 12, 1559-

Lerch, K. (1978) Proc. Natl. Acad. Sci. U. S. A . 75, 3635-3639 Horowitz, N. H., Fling, M., Macleod, H., and Watanabe, Y. (1961)

Cold Spring Harbor Symp. Quant. Biol. 26,233-238 Sussman, A. S. (1961) Arch. Biochem. Biophys. 45,407-415 Horowitz, N. H., and Fling, M. (1953) Genetics 38,360-374 Lerch, K., Longoni, C., and Jordi, E. (1982) J. Biol. Chem. 257,

Lerch, K. (1982) J. Biol. Chem. 257,6414-6419 Katan, T., Arnon, R., and Galun, E. (1975) Eur. J. Biochem. 59,

Pace, C. N. (1975) CRC Crit. Reo. Biochem. 3, 1-43 Aune, K., and Tanford, C. (1969) Biochemistry 8,4586-4590 Forman, H. J., and Fridovich, I. (1973) J. Biol. Chem. 248,2645-

Feder, J., Garrett, L. R., and Wildi, B. S. (1971) Biochemistry 10,

Fersht, A. R. (1971) Cold Spring Harbor Symp. Quant. Biol. 36,

Nevo, E. (1978) Theor. Popul. Biol. 13, 121-177 Milkman, R. (1973) Science 182, 1024-1026 Selander, R. K., and Levine, B. R. (1980) Science 210,545-547 Kaufmann, F. (1966) The Bacteriology of the Enterobacteriaceae,

Wilson, A. C., Carlson, S. S., While, T. J. (1977) Ann. Reu.

Lerch, K. (1976) FEBS Lett. 69, 157-160 Hermodson, M. A., Ericsson, L. H., Titani, K., Neurath, H., and

Lerch, K., and Fischer, E. H. (1975) Biochemistry 14,2009-2014 Horowitz, N. H., Fling, M., Horn, G. (1970) Methods Enzymol.

Riiegg, C., and Lerch, K. (1981) Biochemistry 20, 1256-1262

681-686

Tratschin, J. D. (1979) Biochemistry 18, 5698-5703

1565

6408-6413

387-394

2649

4552-4556

71-73

Munksgaard, Copenhagen

Biochem. 46,573-639

Walsh, K. A. (1972) Biochemistry 11,4493-4502

17A, 615-620

K. Lerch, unpublished data.

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Tyrosinase Isoenzymes: Primary Structure and Thermostability 6425

3.0 (11 2.0 1.9 (2) 2 1 0.3 ( I ) 1.0

1.0 ( 1 ) 1 . 1 1.0 (1) 1.0 * z I*) 4.9 7 . 2 ( 2 ) 2 . 2 1 1 (1) 0.6

1.4 ( 1 ) 1 . 1 1.0 (1) 1 . 1 2 . 2 ( 2 ) 2 . 2 i .0 (1) 1 , i.1 ( 1 ) 1 . 1

1.0 (11 1.0

1.0 (1) 1.0 1.1 ( 5 ) 5.0 0.9 ( 1 ) 1 . 0 0.8 (1) 0.9 0.9 (1) 0.9

1.7 ( 2 ) 2.1 1.0 (1 ) 1.11 1.1 (1) 1.1

7.0 ( 7 ) 1.0

1.1 (I) 1 . 1

1.8 I21 2.0

0.9 (1) 1.0 .o 1 . 0 (11 1.0 1.0 (I) 1.0

*,m CB1-TI c

2.0 (21 2.0 2.4 (21 2.1 1.1 ( 1 ) 1.1 (11 1 1 I O (11 1.1 1.0 (I) I.,

0 8 (11 0.6 3.1 (31 1.1 1.0 ( 1 ) 1 .2

1.0 (1) 1.1

1.0 ( 1 ) 1.0 1.1 11) 1.0

1.0 ( I ) 1.0 1.1 (1) 1.0 2 6 (I1 1.0 1.0 ( 0 1.0 1 0 (1) 1.0

1 . 2 11) 1.1 1.1 ( 2 ) 1 .0, 1.1 ( I ) 1.0 0.8 (1 ) 0.9

1 n # > I " 0

1.1 (1) 1.1 1.1 ( 1 ) 1.2

, , " . ~ 1.1 (1) 1 . 1 1.0 (1) 1.0

0.7 (11 0 9 0.9 ( 1 ) 0 8

1 0 (1) 0.9 1.0 / I ) 1.0 3 . 0 (31 3.1

I , , 1 " 1.0 I l l 1.1 1.0 (11 1.0 1 . 0 ( 1 ) 1.0 1.0 (1) 1.0 1.0 (1) 1.0 1.0 (1) 1.0

+ I l l +

2 2 121 2.0 IO) 1.0

"dl 1.0 11) 1.0 1.1 Ill 1.1

1 1 (11 0 P

. 5 T 97 - 100

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6426 Tyrosinase Isoenzymes: Primary Structure and Thermostability

i)ll,lo CW-Thl CW-Th2 C84-Th3

1s '?? Sing TS TL Sing TS TL 81ng fran from

AID 2.0 (21 2.0

ser lhr

Gl" 1.0 ( I , 1.0 1 0 ( 1 ) 1.0

Vm 1.0 (11 1.0 G ~ Y ula Val

Leu 1.0 ( 1 ) 1.0 1.0 11) 1.0 1.1 ( 1 ) 1.0 Ile

r y r vhe nlr 2.1 (21 2.0

Ar9 LYS

Trv + (11 * Y e 0.840 0,825 0.401 0 . 4 M 0.0% 0.WO Ve,Tl 0.840 0.400 0 071 Residue> 301 - 306 312 - 314 319 - 323

TABLE X I - Sequence a m l y r l i o f CB1 frm s t r a i n 1s (250 r m 1 C l )

Cycle P l H - M Yleld I d e n t i f l c a t l o n lrnll

170 HVLC

150 R 205 HPLC 125 HVLC 195 HVLC 107 HPLC 115 HRC 60 HVLC 85 HVLC 90 HVLC 70 HPLC

30 HVLC, R so HVLC

40 HVLC. n

200 m c

50 w L c

IO nvLc. R 21 n u . R

20 nvtc. R 6 nvLc

25 nvLc

2 s w L c 12 nvtc

10 HPLC, R 25 RPLC. R

25 HVLC 6 HPLC

30 HPLC 6 HVLC

I Thr 2 Pro 3 Arg 4 Pro 5 u l a 5 Pro

8 sei 7 i y r

I O Phe 9 l h r

I 1 V a l 12 Ala 13 G l n 14 Glu 15 G l Y

17 Ser I 6 Glu

18 Gl" 19 ser 20 Gln

22 Thr 23 Pro

28 Glu 24 L w

26 Pro 27 Phe 28 Trp

21 E

29 IlP 30 Lyr

in I 50 R 40 R

4U GLC 10 GLC

30 GLC bo R on D ," . 50 GLC,R

1 w GLC 60 GLC 70 GLC,TLC 60 GLC 40 GLC.R 50 GLC.R 30 R 60 GLC 20 R so HVLC

30 R 50 GLC.R 20 GLC

40 GLC 20 7LC.R

30 GLC 15 1LC.R 15 TLC,R IO R

no GLC,R

30 nvic

I 2 Ala 5 GLC

TABLE XYI- Utwted rwuence a m l y r l r O f C U fmn . t l d i " S , * (6W I.Dl.1I

cycle nn-m v d d rdemf (ca twn lm1 I

1 ser 1 w GLC.R

3 Leu 210 GLC 2 Ser 6 0 GLC,R

4 Gl" 2w GLC

6 Ser 100 GLC 5 Val 320 GLC

7 U l a 420 G L C 8 Vhe 190 GLC 9 LIP 70 GLC.R.TLC

IO vro 1m GLC 11 Le" a3 GLC 12 Phe 250 GLC 1 1 Trp 70 GLC 14 Leu 180 GLC

16 HIS 20 R 17 Val 80 GLC 18 As" 30 R . TLC 19 Yal 70 GLC 20 Asp 40 6LC.R.TLC 21 Arg 10 R 22 Leu SO GLC 23 lrp 10 GLC.1LC 24 ser 20 R 25 l l e 10 R 26 l r p 5 R,TLC 27 Gln 2 m c 28 Asp 6 HPLC 29 Leu 4 HRC 30 A m 1 n n c 11 P r n 6 nPLc

1 5 nir 20 I

1 G l u 2 L e u

4 G i n 3 6,"

6 T y r 5 A m

8 Gl" 7 Pro

IO Phe 9 Gin

11 as" l Z Le,

40 1 06 43 37 31 30 19 16 22 1 3 14 10

8eg"D"CL 4rp-Scr-lhr-Thr-Vhe-Gly-l~-~l l"lyr-Pro

h i m l i l d i : Ar~.Ser.lhr.PDe.Gly.iyr.Ala,Prn.Gli.Lyi Cnpar l t~on: 7.1 1.1 216 1.0 1.0 1 6 1.0 1.1 1 . 9 0.9 E W " d e g l d d a t m

G l u - T h r - G - L y l

step 1. 0.a 0.9 3.0 1.1 1 . 2 1.8 1.0 1.1 2.2 0.9

r t e p l - 0 . 3 ~ ~ 0 9 l 1 . 0 1 . S 0 . 9 O 8 i P 0 . 7 step 1: 0.1 0 . 5 2.7 1.0 1 1 1 . 8 1.1 0.9 2.0 0.7

1 vhe 20 HVLC 2 Thr 4 HPLC.R

4 "15 10 I 8 v m 6 Val 11 HVLC

7 HVLC

7 A m 5 Pro

5 HPLC

3 24 nvLc

5 m c

2

8 9

10 11 12 13

31 32 Ala 18 HPLC

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C Rüegg, D Ammer and K Lerchwild type strains of Neurospora crassa.

Comparison of amino acid sequence and thermostability of tyrosinase from three

1982, 257:6420-6426.J. Biol. Chem. 

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