Amino acid composition and thermal stability of proteins

Amino acid composition and thermal stability of proteins P. K. Ponnuswamy, R. Muthusamy and P. Manavalan DeparlmeJTI O/ Pllysics. .4ttlom)mous Post qraduale ('etltre, 7h'uchirapalli (Received 17 August 1981)

620 02(), T~m~ihmdu, h~dia

This stmlv im'esti:lates the rehttimtslTip between /he thermal stability q l a olohtdar proteiH amt it.s amim) acid compo.sitio~l. 71~emethod deals ~'iI]7 the rehttioJt.shil~ hetweet~ the amino acid eompositiotts and meltitttl points in a ~et ~?lproleiits hy eOml~utitl:l sin~lh'-rcsidlte aitd ~lm)ttp eorrehtti(ms. Groups ~?[residues are showH to stabilize or destahilize the molecule aoaitTst teml~eVatm'e. The stabiliziml oroup eoltsisls (71 l~ohtr-ehar:led residues and mml)ohtr residues possessil~ 0 hi:lh sm'rottndiH 0 hydrophobieit.v. The i~ohtr-tmehar~ted residues destabilize the molecule a~lainst temperature, serine heinq the most destahiliziJly residue. A cery hiqh cooperatit~it y exists amon# :he stabilizin# nonpolar residues suq,qestin# that their characteristic clustering inside the globule may enhance the :hermostahility of a protein. In small ,qlohular proteins which act as single cooperative units, the melon# temperature remains mainly a /imction olamino acid composition, whereas in complex molecules it depends on otherl~tctors als~J. Keywords:

Proteins: globular proteins: thermostability; residue cooperativities

Introduction It is well known that the function of a protein molecule is related to its conformation under physiological conditions. It has been proved from denaturation studies on proteins ~ 3 that a change in environmental conditions (such as pH. temperature, etc.) affects the stability and hence the function of the protein molecule. The thermal stability of a protein molecule deserves particular attention since proteins originating from thermophilic organisms are noted to be stable even at high temperatures which can cause denaturation in other macromolecules ~. No explanation for this obserw~tion is at present available. In addition, thermophilic species are thought to be the oldest forms of life s 7. Hence a systematic study on the thermal stability of proteins will allow a better understanding of how organisms have emerged and evolved. Koffler s and Bigelow ~ suggested that thermal stability might be attributed to the more numerous and stronger hydrogen bonds or hydrophobic bonds in thermophilic proteins compared with their mesophilic counterparts. The importance of hydrophobic bonds in determining protein stability was stressed since their strength is supposed to increase with temperature tip to ~ 60 70 C, whereas the contribution of hydrogen bonding to the free energy is said to remain more or less constant with increasing temperature 1°'~. This favours the idea that hydrophobic force is the dominant factor in the thermal stability of proteins. However, an extensive comparison of the proportions of hydrophobic residues in thermophilic and in mesophilic proteins has not revealed any such definite relationship between hydrophobicity and thermostability ~~~-'~3 Bull and Breese ~4 studied the relationship between the melting point of a protein and its physical properties, such as the average residue volume and the average hydrophobic index, and suggested that hydrophobic residues actually destabilize the molecule with temperature. Yutani eta/. 1s suggested that stability of an enzyme might be greatly enhanced by increased hydrophobicity through substitution of a few suitable 0141 8130,82'030186 05503.00 ©1982 Butlerworth & Co. tPublishers Lid

186

Int. J. Biol. Macromol., 1982, Vol 4, April

amino acid residues. According to Stellwagen and Wilgus 16 protein thermostability need not depend on the average residue volume, but the ratio of surface area to volume for a given protein domain or subunit is a critical factor of thermal stability. Recently, the thermal stability of proteins has been studied using the available sequence data of thermophilic and mesophilic proteins by Argos et al. ~7, and Ikai 18 has proposed that the aliphatic index has a better qualitative correlation with the thermostability of globular proteins. Hence, due to controversial views and lack of good quantitative interpretation regarding the thermal stability of proteins, we have undertaken a systematic study of this problem. It has been suggested that the specific three dimensional structure of a protein molecule is mainly governed by residue residue cooperativities in the molecule, which cause specific types of residues to cluster together with characteristic environments around each type of residue ~. Kuramitsu 2° has also pointed out that such cooperativities play an important role in retaining the characteristic biological activities of the protein molecules, aspartokinase and threonine deaminase even after a considerable increase in the surrounding temperature. Thus, it seems that specific cooperativities among w~rious residues determine the increased or decreased stability of protein molecules with temperature. In this article we present the results of our study to establish the stabilizing and destabilizing groups of residues by correlation. We use the melting points of proteins and their amino acid contents as primary sources t\)r our investigation, since the melting point is tile parameter directly related to heat stability.

Experimental Relationship between melting poillt and amino acid eorilellt ~l a proteill

Proteins used for the investigations were: bovine pancreatic trypsinogen, bovine pancreatic chymotrypsinogen, porcine pancreatic elastase, swine

Thermal stability of proteins: P. K. Ponm4swamy et al. pepsinogen, bovine pancreatic ribonuclease, bovine pancreatic carboxypeptidase, horse liver alcohol dehydrogenase, bovine erythrocyte haemoglobin, egg white lysozyme, bovine insulin, soybean trypsin inhibitor, sperm whale myoglobin, bovine ~-lactalbumin, horse heart cytochrome c and bovine milk fl-lactoglobulin. The amino acid compositions for these proteins have been taken from Dayhoff 2. and from Shotton et al. '-2 The melting points were taken from Bull and Breese ~'~ and Stellwagen et al.~ ~'

Sin,qle residue correlation The percentage compositions of each of the twenty different residues for each of the fifteen proteins were computed. The influence of each type of residue on the melting point of the protein molecule was determined by calculating the correlation between them: i'=

NZXY-(YX.ZY) 1

(1)

{ [ S E X 2 - { Y X ) 2 ] [ N z },2 __ (y y)2]} 2

where r is the residue correlation coefficient: N, X and Y represent, respectively, the number of proteins, the independent variable (percentage composition of a residue) and the dependent variable (melting point).

Group correlation The group of residues which might stabilize or destabilize the molecule can be determined from the single residue correlation coefficients. For this purpose the twenty amino acid residues were classified into two groups. The classification was made on the assumption that the subgroup of residues to be obtained from these classified groups must either stabilize or destabilize the protein molecule. G r o u p I consists of residues corresponding to the first fourteen highest r values and group II consists of residues corresponding to the first fourteen lowest r values. In each of these two groups all of the fourteen residues may or may not contribute to the stabilization/destabilization of the molecule and only a selected subset of residues within the group could stabilize or destabilize the molecule. In order to find this subset of residues, all possible combinations among the residues of each group were considered using the formula n!/[ln-p)!p!] where n is the total number of residues in each group and p is the number of residues taken at a time, which varies from 1 to 14. For each combination of residues, the sum of the respective percentage compositions (X) was found for each protein and the respective correlation coefficients between the variables X and Y(melting point) calculated using equation (1)ithus, we have fourteen r values for p = 1, ninety-one r values for p = 2, etc.). From the set o f t values thus computed for each of the two groups, the subgroup of residues that have the highest positive correlation coefficient and the subgroup that has the highest negative correlation coefficient were selected for analysis. Multiple correlation The melting point of a protein is dependent both on the stabilizing and the destabilizing groups of residues and hence it is appropriate to consider the melting point as a function of these two groups of residues. This is done by the use of the multiple regression technique; the multiple

correlation coefficient (Rt and the regression equation were determined by standard procedures 23. Results and discussion

Stahilizim, i and destahilizimt residue ~lroup.~ The single residue correlation coefficients for the twenty types of residues calculated using equation (11 are givcn in Table 1. This Table shows that there is a wide distribution of r values having both positive and negative signs: Glu has the highest positive value and Ser has the highest negative value; Lys and Leu arc the other two residues which show recognizable positive correlation and Val is the only other residue showing noticeable negative correlation: the values of the rest of the residues are v c o low. Hence a meaningful interpretation of these single residue correlation coefficients is not possible. However, the r values given in T, hle I can be used to select sets of stabilizing and destabilizing residues. The stabilizing set (group I) is taken to be the fourteen residues from the top of Table 1, and the destabilizing set (group ii) being the fourteen residues from the bottom of this Table. Using these two groups of residues, correlations between the melting point and the amino acid composition have been computed for all possible residue combinations. The computed results show that a number of combinations of residues in group I have correlation coefficients above 0.90. Similarly, some specific combinations of residues in group I I show high negative correlations. O[" the 14 residues of group I, the combination (X ~} of residues Asp, Cys, Glu, Lys, Leu, Arg, Trp and Tyr has the highest positive correlation coefficient 1r-0.98221 and the combination (X2) of residues Ala, Asp, Gly, Gin, Ser, Thr, Val and Tyr from group 11, gives the highest negative correlation coefficient ( r - - 0 . 9 7 5 2 1 . The least square lines relating the melting point with X1 and with X2 are, respectively, given as: }' = 11.67367 + 1.52644 X l and Y= 135.37432- 1.34938 X_,

{3)

Table 1 Single residue correlations with melting points in globular proteins Residue

Correlation coefficient It)

Glu Lys Leu Phe His I le Asp Cys Met Arg Asn Tyr Trp Ala Pro Gin Gly Thr Val Set

0.8469 0.5776 {).4627 {).2827 {).2607 0.1543 {).1427 0.0516 0.0452 -0.0267 -0.0874 - 0.1159 -0.1180 -0.1753 -0.1797 - 0.1864 0.3418 {).3742 - 0.5766 -0.8687

Int. J. Biol. Macromol., 1982, Vol 4, April

187

l h e r m a l .,;tahilitv ql l,'otein.s. P. K. l)omnl.,;wanly el al. 7uhle 2 gives the estimated melting points using the above regression equation along with the corresponding experimental ,,'+titles for the 15 proteins. The standard error of estimate for the predicted rabies is 1.4559. The agreement between the predicted aim experimental rcsulls is, thus, reasonably good. ('onsideling the l-l+tture of lesiducs constituting the positive and negative earlelation groups, it is illtercsting to note that the residues which stabilize the molecule arc mainly charged (Asp. Glu, kys and Arg) and nonpolal(('ys, Leu, Trp and Tyr) ones. It is WOltla pointing out the high surrounding hydrophobicity associated with the latter residues, i n t h c r a n g e 13 15 k c a l m o l ].24,e> Also notice that most of these stabilizing residues are helix formers. It is again interesting to note that all the polarneutral and ,,,hart (Ser, Thr, Gila and Gly), and the compact and short nonpolar (Val :.llld Ala) residues belong to the destabilizing category. It is surprising to scc that Asp and Tyr occur in both the groups: this lnay be duc to the dual role played by these residues: these two residues characteristically appear both in the interiol ~md on the surface of the molecule. The residues F'hc, lie, Met, His, Asn aim Pro seem to bc indifferelat +is they do not belong either to the stabilizing or to the destabilizing groups. These re~,tllts suggest that there are specific coopcrati~ities a m o n g specific kinds of residues which could either elevate the tnelting point of a proteila or decrease it depending upon their proportion in the total composition.

90

o/

85

o:/

80 el

/

75 0

E

70

oa

g

o

65

o

5

60

55

50

0

25

a

I

t

I

I

I

55

4o

45

50

55

O/o Composition of stabilizing group ~ X I

90

]Jt'oct'dttFe le.st The validity of the procedure was tested by redetermining the coefficients m equation (41 and omitting one protein at a time in the calcuhttion. The coefficients thus c o m p u t e d can be Used to predict the melting point of the olnitted protein. Table 3 lists the predicted melting points, the relevant coefficients, the experimental melting points, and the difference between the observed and

85

80

O

o

oo

~E r 5

ro ff 2 Experimentally observed +,+lad theoretically predicted inching points for fifteen globular proteins

fable

65

o

Mehing points (71,,11 (')

60 Protein"

55 5(

b

50

3i5 %

410

4i5

I

50

I

5J5

Composition of destabilizing

group,

60

65

X2

F i g u r e I ta) Plot of the melting temperature (11,,) against stabilizing group (X t ) of residues in 15 globular proteins. (b) Plot of the melting temperature (T,,,) against destabilizing group (+k2I of residues in 15 globular proteins

Plots (a) and (b) in Figtu'e ] show the theoretical lilacs for the above two equations. The experimental points fall almost on the respective theoretical lines. The multiple COl+relation coefficient obtained by nsing both X 1 and X, :is independent variables is 0.9907 and the corresponding lCglessiol~ equation is: } = 64.46202 q 0.89432 .V~

!8~]

0.59065 X 2

Int. J. Biol. Macromol., 1982, Vo[ 4, April

(4)

Trypsinogcn I [ ; ) ('hymotrypsinogen(fi) Elastase(fi) Pepsinogen Ribonucleasc(24-/7) CarboxypeptidasclTfi Alcohol dehydrogenase I;~ [h Haemoglobin (ml Lysozyme(7+//t Insulin (~q fl') Trypsin inhibitor Myoglobin 171 :~-Lactalbumm 1~+/;) ('ytochromc c I:~q [i) /']-Lactoglo bulin

Nati;'c molecule

Expertmental;'

Calculated

Monomer Monomer Monomer Monomer Monomer Monomer

55 57 57 60 62 63

56.96 57.61 55.22 58.97 61.38 63.68

Tetramer Tctramer Monomer Hexamer Monomer Monomer Monomer Monomer Dimer

64 67 72 76 77 79 83 83 H3

64.97 67.77 72.58 76.39 74.25 79.48 84.77 ~0.72 83.1S

" The structural type is noted in brackets ~' Experimcnlal vah.ies were taken from Bull and Stellwagen c't a/. ~+'

B r c e s c 14

and from

Thermal stahility (?lproteins: P, K. Pomu~swamy et al. Table 3

Tcst of thc predicted melting points for the proteins Constant

Protein

Coefficients

Mehing pain( (7;,,1 I C)

in

Trypsinogen Chymolrypsinogcn Elastasc Pepsi l]OgCl] R ibonuclease ('arboxypcptidasc Alcohol dehydrogenase Haemoglobin Lysozyme I nsulill Trypsin inhibitor Myoglobin z-Eat(albumin ('ytochrome c /#Lactoglobulin

equations

X

X,

Predicted"

Experimental

Error

65.63 63.58 64.67 70.70 63.46 66.04 74.11 62.66 63.56 63.23 63.60 67.(16 62.64 55.72 63.44

0.865 0.901 0.908 O.X25 0.909 0.873 0.774 0.916 0.907 0.912 0.894 0.867 0.939 0.979

-0.589 -0.577 - 0.608 0.667 -0.583 0.605 0.692 0.570 0.581 0.578 0.577 0.622 - 0.5b;5

57.37 57.74 54.78 5~.53 61.30 63.78 65.60 67.87 72.64 76.48 74.00 79.67 85.34

55 57 57

-2.37 -0.74 + 2.22 + 1.47 4 0.70 - 0.7X 1.60 (I.87 0.64 0.4S 4 3.00 -0.67 - 2.34

0.4~2

~0.04

X3

4 2.96

0.909

0.581

83.27

83

-(I.27

O0

62 63 64 67 72 76 77 79 ,';3

Predicted by substituting the constant and the coefl'icicntsof the 'variables ,\'~ and X 2 using equation (4). Thc parameters in columns 2, 3 and 4 ~crc obtained without including the respective protein primarily equation (4)(as it gives the more accurate wtlue 85

O

of T,,,)for predicting the melting points of globular

proteins with the other additional below.

80

75 g .o "0

7 0

Q.

5

6~

o

~_e 60

0

55 50

50

5~5

6;

6; 70 715 Tm (°C) Experimental

8'0

815

Figure 2 Plot of the predicted melting temperatures rer.~us the experimental observations. The line at the slope 45 corresponds to the perfect agreement between theoretical and experimental values

predicted melting points (errors}. The predicted mclling points are plotted against the experimental T,, wtlues in Fiqure 2. The line drawn with a 45 slope wouM represent a perfect agreement between the predicted and observed melting points and we notice a very good agreement. This lest, although not so exacting due to the a m o u n t of data, provides confidence in the present type of statistical study.

Lecel e l conlidenee We calculated the limits for equations (2), (3) and (4) and found the T,,,, values corresponding to 10()':,, stabilizing residucs to be: 164.3, 135.4 and 153.9 C, and 11.67. 0.44 and 5.40 C, respectively, corresponding to the presence of 100",, destabilizing members. Although these values seem improbable, since amino acid residues in a protein molecule are proportionately distributed into stabilizing a n d d e s t a b i l i z i n g groups, w e can use e q u a t i o n s (2), (3) and

factors described

More complex proteins We examined a few other complex proteins not included in the study so Dr described, and found that for 10 such proteins, viz. subtilisin, avidin, superoxidc dismutase, fi-galactosidase, serum albtunin, concanava!in A. catalase, aldolase, bovine carbonic anhydrase and c y t o c h r o m e c peroxidase, the deviations of the predictcd T,,,, values from the respective experimental ones were large. It may be due to critical factors such as the size of domains and polymerization, ligation of strongly bound metal ions, cofaclors, packing densities and intrasurfacc interactions its indicated by Stellwagen el al. ~° X-ray investigations s h o ~ that the corc of subtilisin e" consists ahnost entirely of packed hydrophobic side chains and, in the absence of disulphide links, this tight packing core is responsible for its thermostability: also in this protein the polypeptide chain is foldcd in thrcc distinct section> which may again reinforce thcrmostabilit 3. As pointed out by Stellwagen el af t". serum albumin is a multidomain protein, aldolase, catalase :,uld /{-galactosidase are large polypeptidc tetramers, and c y t o c h r o m e c peroxidasc e~ is folded into t ~ o clearly defined domains and thus the denaturation process in thcsc proteins seems to be me)re complcx. In the case of avidin 2s. the presence of the c a r b o h y d r a t e moiety in the molecule may provide additional stability. On binding metal ions, the stability of supcroxide dismutase (dimer x~itla Cu and Zn ionsl and concanavalin A (tetramer with Mn and Ca ionsl ~ o u l d increase and hence these two proteins h a \ e higher melting temperatures in spite of their ha~ing smallcr a m o u n t s of stabilizing typc residues. The prescncc of Zn ion in cltrbonic anhydrase has bOllle inllucncc on the cooperative transition of the molecule. For the complex proteins discussed abe',e, equation ~4) may not bc applicable its a predicting device. Par the protcins: alcohol dehydrogenase, haemoglobin, insulin and carboxypcptidase, the experimental 71, values agree with our predicted resu[ts and hence thc denaturation proccss

Int. J. Biol. Macromol., 19S2, Vol 4, April

189

Thermal stability olproteins:

P. K. P o n n u s w a m v el al.

of these protein molecules must bc of a cooperative nal.ure.

O u r results so far discussed suggesl that the melting temperature is mainly a function of the anlino acid composition m small c o m p a c t globular proteins which act as single cooperatix, e units. We suggest that in the case of small globular proteins which undergo cooperatlvc thermal denaturation, equation /4) can be used for predicting their melting tcmpcratures. ~'Oltlt' ~lc'tlt'rdl poinl,s

tl) Tile results so far described were obtaincd without including effects due to haem moieties and disulphide links a h h o u g h they were present in a few proteins. However, scrutiny of the amino acid residues themselves has provided vahiable information regarding the thermal stability of small globular protein molecules, and a very good agreemcnl between the prcdicted melting points and tile experimental results has been found. This fact mdicates that the effect from the haem moieties and distilphide links may be small c o m p a r e d with the effects troll amino acid composition, metal ions and polymerization. It is to be pointed out here, the suggestion made by Singleton and Amelunxen a that the capacity for cross linking distllphidc mteractions may be reduced in the thermally stable proteins. There arc fewcr sulphide links in haemoglobin and c y t o c h r o m e c 12 and 11 than in the other proteins investigated, while myoglobin has no ~,ulphidc bridge; this suggests that the sulphide links and haem moieties complement each other as far as the stability of the protein molecule is considered. t2) To support our present results, we point OUt the expcrimental results obtained by H o w a r t h >~ that the rcsidues Ala, Asia, Thr, Tyr and Set take part in the unfolding process early in the thermal denaturation of RNase, whereas Ile, Mct, Phe and ttis come into play about 5 10 (7 abo\,c the main transition temperature T,,,. From this it is evident that the residues undergoing early denaturation are all {except Asn) destabilizing ones found in g r o u p I1. It also sccms that indifl'erent residues may undergo denaturation at a temperature even above the nlelting poinl. i3t Since the proteins tlsed in the present inx,cstigation arc nlostly simple monomers, ~-type m o n o m e r s with a haem g r o u p and dimers without metal ions, and the proteins disagreeing ~ith our regression analysis arc mostly fl-sheet proleins with strongly bound metal ions and large polypeptide telramers, it is inferred that the intluencc of metal ions binding with fi-sheet proteins is sonlewhat greater when compared with :c :~/] or l:z 7-fi)ty, pc proteins. t4) As pancreatic trypsin inhibitor and ferrcdoxin arc small compacl globular proteins, we predicted their melting points using equation 141. The predicted 71, values for PTI, thcrmophilic ferredoxin and mesophilic fcrredoxin arc: 81.1, 63.7 and 57.7 C, respectively. Even though Aplq,AT discontinuity could n o t bc observed ~' for PTI at temperaturcs tip to 98 ('. the above predicted 7],, xalue may bc very near to tile corrcct melting lcmpcrature of this protein. It is again interesting to note that tile predicted 71,, values corresponding to tile thermophilic and mesophilic ferredoxins indicate the reliability of the relationship betw ten nlelting tcnlpcraturc and amino acid composition in protein molecules: thermophilic ferredoxins lost only 5 to 10"<, of lhcir aclivity upon heating at 70 (" for 1 h, ;vllcrcas the

190

hat. J. Biol. Macromol., 1982, Vol 4, April

mesophilic proteins lost 70 to 75".> of their activity under the same conditions 4. 15) It is noted from the restilts that the thermal stability comes fi'om both the polar-charged and tile nonpolar residucs, while tile uncharged polar residues, in contrast, destabilize the molecule. A significant observation is that out of four stabilizing n o n p o l a r residues, three ICys, Leu, Trpl are associated with high surrounding hydrophobicity 2"*-'s and thrcc polar rcsiducs IAsp, Glu. L x,s) are charged, ttence mediuln-rangc and tong-range interactions {electrostatic interactions by charged rcsidues) play an important role in maintaining tile structural integrity of a protein molecule against temperature. It is noteworthy to point out here the fact that the long-range van der W a a l s / h y d r o p h o b i c interactions providc stability against disturbing external forces and conformational fluctuations s~. The thermal stability of a protein thus could nol be accounted by a single type of force alone but forces such as ion ion interaction, hydrophobic clustcring, etc., play competing roles31.

Acknowledgement This work was supported by a research grant to P.K.P. flom the Department of Science and Technology, G o v e r n m e n t of India. References I

Pohl, F. M. qn#eu, hltl. Edn. 1972, I1, 894

2 3 4 5 6

Tanlord. ('., Aunc, K. C. and lkai, A. J. Mol. Biol. 1973, 73, 185 Burgess.A. W, and Scheraga, H. A J, 'lheor. Biol. 1975,53, 403 Singleton.R. and Amelunxcn, R. E. Blot. Rer. 1973, 37, 320 Brock,T. D. and Darlands, G. K. Scicm'e 1970. 158. 1012 I'anaka. M., Haniu, M.. Maisueda. (J.. Yasunobu, K., ttimes, R. H, Akagi..I.M., Barnes, E. M. and l)cvanathan, T. J, Bi,l. ('hem

7 8 9 I() 11 12 13 14 15 16 17

18 19 20 21 22 23 24

1971. 246, 3593 Peck. H. I). in "('t)lll'I Extreme En,¢ifcln,', Amer. Resell ch Centre. NASA, Mc)['['ctl Field, Ccili., 1972, pp. 26 28 Kofllcr. 1t. Ba~l. Rot. 1957. 21. 227 Bigclou. ( ' ('. J. lheor. Biol. 1967, 16. 187 Scheraga. H. A. in "['he Proteins" (Ed. H. Ncurath) Academic Press. New York, 1963, Vol. 1, pp. 477 594 Brandls, ,I F. in "Thermobiolog_C (f'd. A t t . Roscl Academic Press, New York, 1967, pp. 25 72 Singleton. R.. Kimmcl. ,I. R and A m e h m x c n . R. t!..l. Bi.t. ('hem I969, 244. 1623 G o l d s a c k , D. E, BioFoh'mcr,s 19711, 9, 247 Bull. tt. B. and Brccse, K. Itch. Biochem. Biophys. 1973, 158, 681 Yutani. K.. O g a s a h a r a , K., Sugino. Y. and Malsushiro, A. Nature 1977, 267, 274 Sicll~agcn. I!. and Wilgu,,,, H. :\,'au~rc 1978, 275. 342 Argos, P.. Roxsmann, M. (;.. G r a m {J. M.. Zubcr, H., Frank, (i.

and Tratschin, J. D. Biochemistry 1979. 18. 5698 [kai, A..I. Bi,~ht',l. 1980. 88, 1895 Mcincl~ahm,P. and Ponnuswanay, P. K .,h'ch. Biochem. IJioph~s. 1977. 184. 476 Kuramitsu.ft. K. Biochim. Biophys. ,,|~ta 1968, 167. 643 Dayhofl',M. O. in 'Atlas of Protein Sequence and Structure', Silver Spring. Maryland, LISA. 1972, 1973. 1976 and 1978 Shotton, 11. M, and HarrieT, B. S. Nature 1970, 225, 802 Blair. M. M. m 'Elementary Slaiti~tics'. ttenry Hold ('o.. Ncw York. Ic,~52 Manawilan.P. and Ponnus'aamy. P. K. :\'aturc 1978, 275, 673

25

P o n n u s w a m y . P. K.. Prabhakaran, M. alld M a n a x a k m . P. F;i,chim. Biophvs, t(ra 1980. 623, 301

26

Blackburn, S, in "Enzyme Structure and Function'. Marcel l)ekkcr, Inc., Nc~v York and Basel, 1976, p. 226 Poulo~,.l L.,t'rccr. S.T,.Aldcn, R.A,,Edv, ards, S . L . Skogland. [ ]., l a k i o , K., [-riksson. B., Xuong, N.+ Yonelani, T. and Kraut,,l.

27

28 29 311 31

,I. Biol. Chem. 1980, 255, 575 l)cLangc, R. ,I J Biol. ('ht',l. 1970, 245. 907 Hov, arih. () W Biochim. Biol:,hv',. 4([(/ 1979, 576, 163 P o l l n u s ~ a l l l } , P. K. and P r a b h a k a r u n , M. Biochem. Bioph,~ Rc.. ( , m m l m . 1980. 97, 1582 M a l h c , a ~ . B . W . . W e a v e r . L. It. and Kester. W. R..I Biol.('hcm 1974. 249. 81llll