ro ®
, vo~ . r7,yp~ rt-8~r. Pmp Ltd. 1979.
oo~i-oioil79pioi-oo~nsozoo~o
Printed la t3reet Hrltain.
INFLUENCE OF pH ON THE KINETIC AND SPECTRAL PROPERTIES OF PHOSPHOLIPASE A2 FROM BITIS GABONICA (GABOON ADDER) SNAKE VENOM CORNELIS C. VIIJOSN
and Dewy P. Borns
Molecular Biochemistry Division, National Q>ennical Research Laboratory, Council for Sci~tific and Industrial Iteeearch, P.O. Hox 395, Pretoria, 0001, Republic of South Africa (dcceptedjor publlaatlon 28 .lpril 1978)
C. C. Vii.Ta~v and D. P. Ho~res . I~ueaoe of pH oa the kinetic and spectral properties of phospholipase A= from Blur gabortka (Ciaboon adder) snake venom. Toxlcon 17, 77-87, 1979.-The i~uenoe of pH on the kinetic parameteas ~Cae and ka,/%b, and the spectral properties of BitiCr gabonica phospholipase A~, showed that the activity of the enzyme is controlled by groups of p% values 6"8 and 9"2. These groups were found to exhibit enthalpies of ionization (nH~ of 7"1 Kcal/mole and 6"1 Keàl/mole respectively which are eharactcristic of nH~ of histidine sad tyrosine residues. Whilo the dissociation constant (%~ for reaction between enzyme and Cap* was affected by pH, the Michaelis constant (%~ of the enzyme for thophoapholipid substrate was found to ba uninfluenced in the pH range tented i.e. pH S"S-A"0. The binding of Ca3+ to tl~ enzyme is inhibited by protonation of a group of p% ca. 6-0. The oH~ of - 1 "6 Kcal/mole cakarlated for tl»s group is is the rango of the values found for carboiryl groups. The various functions of nucleophi~e, proton donor and Ca3* binding site may be aselgried to the side chains of atructuraliy invariant residues which for the B. gabo,dca plioapliolipase As are located at His-45, Tyt-25 and Asp~6 respectively" Non~ompetltive inhibition of the H+ was obe~ved with respect to both Ca3* and lecithin wlüch probabiy involves an effect of theproton oa feeo ~and theinterconveraioa of theoeatraloomplexee. iIVTRODUCTiON
studies on snake venom phospholipase Az (EC 3.1.1 .4) have shown that the enzyme follows a kinetic mechanism of the ordered bi-ten type (W~.rs, 1972 ; VIIJOSN et al., 1974). Also group specific chemical modification inferred certain residues as funotionally important, either in substrate binding, or the catalytic process itself (W1u.1s, 1973 ; Vu row et x1.,1977) . Wells, from an evaluation ofthe pH dependence of the reaction, concluded that a group with a pK value of 7"6 was involved in the catalytic step of the reaction catalysed by Crotalus adamanteus phospholipase Az ~ "" B[.TS, 1972 ; 1973 ; 1974x). Previous studies on the influence of pH on the rate of the hydrolytic reaction catalysed by Bltis gabonlca phospholipase Az was in accord with Wells' finding (VII.TOSrr et al., 1974). A more detailed investigation of the variation of the kinetic parameters of this enzyme with pH however, did not confirm the previous results. The findings of this study are reported here. PRBVIOUS
MoteHaLr
MATR.RTAi c AND METHODS
Dicaproyl-t-ao-ledthin wan obtained from Applied Science Laboratories (USA) and caproic acid wan a product of Fsstman Organic Chemicals (USA). h?hospholipase As purified from the venom of B . gabonica as described (Hares end Vn row, 1974x) was used as enzyme source . Fnzymo ooaceatration was determined from absorbanee values and e= -2"52 x 10 " M- ~cm_r (V>t.roert, 1974, Ph.D. Thesis, Read Afrikaans University, Johannesburg, Republic of South Afrka).
78
CORNELIS C. VIIJOEN aad DAV1~ P. BOTES
Kfxetlc detamfnxtion Activity ofphsopholipase As was mesa~ued by the acIdimetric sassy described previously (VII.AOBN et ol., 1974) sad modified as follows. The reaction was carried out is a Radiometer TTA31 titration assembly connected via a C3I{2321C combined glass sad reietence electrode to a Radiometer 26 ~rpanded scale pH meter. Liberation of five fatty acid was monitored continuously on a Bechmaa Tea Inch Pot~tiometric Recorder. Recorder response was standardised with 10'sM caproic acid. Full~cale sensitivity of 0"2 pH units sad a Chart epeod of 1-10 in/min wale used. Moaaureo>~ta were obtained in theamostated reaction mixtures of 2 ml total vofime under a constant stroam of nitrogen. Reaction mixtures contained 0"15M KCl and CaQs and dkaproyllecithin at the apatifled concentrations. All concentrations of substrate used is the exPerlmeats reported here, were below the critical micelle oonoentration of 0"01 M (ROHOLT and Scn~owrrz, 1%1). A minimum enzyme molecular weight of 13347 (Bo~aa sad Vn wo~r, 1974x) was used to eapr~ess reaction rates in turnovea numbeaa . Malysis ojkixetlc and spectral data The nom~clature of this paper is that of C~Nn (1963) . Initial velocity data wem first analysed graphically in double reciprocal plots of initial velodty/substrate concentration to check linearity and pattern of the plots. Data conforming to equilibrium ordered initial velocity were ßtted to equation 1. YulB Kb.! -I- .!B -Igtagb
v ~
Y
( C ~ ~`1 -f- H/g~ ~- Ks/H)
~
Y
i2) (3)
- ~ \ 1 -I- H/g~ /
In equation 1, v, V,, Z~ sad Ky have the meanings described previously, v and V, reprneeating the observed and maximum velocities, respectively while K~ is the dissociation constant for substrate A sad S~, the Michselis constant for substrate B (VII.~osrr et al., 1974). pH profiles wero fitted either to equation 2 or equation 3 whero K, and 8s represent dissociation constants of groups that moat be deprotonated or protonaied respectively, for activity, H is the hydrogen-ion oonoeatration, and C is the value of Y, the pH dependent ]vnetic parameter, at the optimum state ofprotonation. Arrhenius plots of (data from spectroscopy studies) were fitted to equation 4 (Rwss~. sad C~xn, 1977), where Y equals pg, T the
pSlT
absolute tempeaature, a -2~,3H03R and b i, a constant. Experimental data were fitted to equations 1-4 by the least aquat+es method deecnôed by CtEUr~ (1967) . The points in the plots indicate exPerimontal data, while the curves wero calculated from ßta to the equations above. SP~~~PY Differraoe spectroscopy were carried out as described (Vn ronrr et x1.,1975) is e Varias Techtron Model 633 double beam spectropimtometer coupled to a Hitachi Model QPD73 recorder, and thermoatated by menas o~f a Lauds Ultra Kryomat Model TK-30-D.
RESULTS The kinetic parameters for the reaction between phoapholipase A2 and dicaproyllecithin were obtained from initial velocity studies carried out as a function of either Cap+ or dicaproyllectithin concentration at changing fixed concentrations of the other substrate . Figure 1 illustrates the results of such an experiment at pH 8"0 and 30°C. Using Caz+ as the variable substrate, a family of straight lines were obtained intersecting to the left of the vertical axis, while a family of plots intersecting on the vertical axis were observed when dicaproyllecithin was used as variable substrate at various fixed Caz+ concentrations. Values for Kr Kb and Vr were calculated from a fit of the data to equation 1. The influence of pH on these parameters wen investigated by performing identical experiments in the pH range 5"5-9"0.
pH ~ects on
B. GaboniQO
2S
60
79
Phwpholipese As
TS
100
Dc~ X~0-2(M~I)
FIO. 1. (A) REC~ROCAL PLOT OF INITIAL VELOCITY Aô A FUNCIiON OP Cas+ WITH DICAPROYIr LSQTHIN (DCL) AS THE st)Ti~ATS.
Reaction was initiated with 14 I+g of an ~zyme solution in distr7led water. The numbers on the right indicato the fixed levels of dicaproyllecithin. Data ware collected at pH 8"0 and 30°C . (B) Reciprocal plot of initial velocity as a function of dicaproyllecithin (DCL) oonoentratioa at various levels of Cas+ . Data were obtained at pH 8"0 and 30°C.
Table 1 compares the kinetic constants obtained for the catalysis by phospholipase Az of the hydrolysis of dipalmitoyllecithin with that of the substrate of the present study. It is evident that only the Michaelis constant and therefore the parameter k~ JKb, were influenced by using dicaproyllecithin as substrate for the enzyme . TAeLE 1. COI~ARDON OF TH8 LII~IIC PARAM611~ OF B. 8abvniao Paa~HpL>PASB AT pH 8"0 AND 2O°C OF DIPALAnT+0YI18CTIId AND DI
$Ubstrate
S;~
As
FoR THS xYDROLYSis
KlnOelC paramDt~ Sb ~
kat
~%b
(min - ') (min-' M-') Dicaproyllxithin 4"6 x 10 - 4 S"6 x 10- ~ 4"8 x lOs 8"77 x 10= Dipabnitoylkcithin' 8 x 10'~ 3"2 x 10- ~ 6"Ol x lOs 1"88 x 10~ 'Values reported previously (VILJOBN et 4.,1974). Ia this publication ~ead%b was incorrectly given as 830 M' l.s'' while it should read 8"3 x 10~ M' l.a'~. The values of k~ , and ~.d%b wero based on a mol . wt of 30,000 and aro for reasons of comparison corrected to the mol. wt of 13347 used in tho present report. t%, represents the dissociation constant for substrate A, %b the Michaolis constant for substrate B .k~,t the catalytic rate constant, obtained by divIdiag V, (maximum velocity) by total enzyme conc~tration, whsle Jfc~,~/%b is the apparent first order rate constant for the reaction of enzyme with substrate at low ooncemtrations of substrate. (M)
COItNELL4 G VIUOEN and DAWIS P. ROTES
3,3
a ô 3,2
0 0
0
0
3,1 0
3,2
0 G
0
0 0
0
2,8
Y
2,4
2,0 5,0
Ei,O
7,0 pH
8,0
9,0
Fw. 2. (Top) Ixnvswce oa pH ox z~ IVlzcae~ corrsr~xr Kb . Data were collexted from experiments similar to those described in Fig. l, at the indicated pH's and a bemparature of 30°C. (Bo~ri~) pH VARTATION OF Kh, nrrreo ~ro ~trszmx 3, pK - 6"4.
Figure 2 shows that pKb is pH independent and a mean value of 5"9 x 10-+M was calculated for this parameter. However, the dissociation constant (K~ for the binding of Caz+ to the enzyme changed with pH (Fig. 2). The experimental data were fitted to equation 3, giving a pK of 6"4. The effect of pH on k~ is depicted in Fig. 3, showing the lxll-shaped characteristics of the profile. The data were therefore fitted to equation 2, resulting in pKvalues of p~,l = 6"75 and p1~Ca,z = 9"1. A similar curve was obtained for the variation of log kq~Kb with pH (Fig. 3). The pK's from a fit of the experimental values to equation 2, were estimated as ply, = 6"7 and p1~s = 9" 3. Values of the kinetic parameter at the optimum pH were calculated from the various fits as k~~ = 8 " 57 x 10~ min -', kajKb = 1"35 x 106 mini M- 1 and K~ = 7"24 x 10- 4M. Examination of the effect of pH on initial velocity, data, demonstrated that the proton acts as a noncompetitive inhibitor with respect to both Caz+ and dicaproyllecithin (Fig. 4). In both instances the family of straight lines obtained when reciprocal initial velocity was plotted against reciprocal substrate at various pH's, intersected to the left of the ordinate, but only in the case of dicaproyllocithin as variable substrate did the lines intersect on the 1/Substrato-axis . It has already been found that binding of Cat+ to the enzyme causes perturbation of tryptophan and tyrosine residues (Vu.~o~r et al., 197 . Figure 5 illustrates this phenomenon at various pH's. From the pH dependence of the change in absorbance at 293 nm at least 3 transition zones could be discerned, centering at about pH 5"6, 6"8 and 9"2 (Fig. 6). The variation of the apparent pK values with temperature is set out in Table 2. These
pH effexts on ~. Gabontex+ Phospholipate As
Sl
6,0
4,8
2,8 ô
2,O
~'~,o
s,o
~,o
e,o
s,o
pH Fix. 3. (Tor) EFFHCr of pH oN lo$ ~OUIKn . The line represents a flt to equation 2, pge , - 6~7 and pKa, - 9"3: pKa + and pl~z rda to the dissociation of groups in free, uncompleAed eazyme . ~HOTPOIN) pH VARrATiON OF l0$ Jkat" Data ware fitted to equation 2, pga+ - fr75 and pSa,s - 9~1 ; pKe,+ and pge,~ mist to the dissociation of groups in the mzyme~ubstrate complex. The parameters lc~ aad k~/Kt, were obtained from initial velocity experiments such as described is Fig. 1, at the indicated pH's . Experiments were carried out at 30°C.
values were fitted to equation 4 to calculate the heat of ionization of groups associated with the various pK values . Enthalpies of ionization of - 1 "6 Kcal/mole, 7" 1 Kcal/mole and 6" 1 Kcal/mole were thus estimated for the groups of pK,, pKs aad pK3 respectively . DISCUSSION
The initial velocity patterns observed in this study showed the diagnostic features for an equilibrium ordered addition of substrates, wherein the addition of Cap+ to the enzyme is followed by addition of lecithin, while the binding of metal to the enzyme is at thermodynamic equilibrium (Cr.rrr ."t~m, 1970). These results confirm those previously obtained using dipahnitoyllecithin as substrate (VtuosN et al., 1974). As can be gathered from Table 1, the long chain lecithins constitute better substrates for the enzyme than the phospholipids containing short chain fatty acid esters . The influence of substrate is not seen on the catalytic constant of the enzyme, since this value is, within experimental error,
CORNELIS C. VILTOEN and DAV1+18 P. HOTPS
82
700
I
0
ô E c É
x
0 600
500
500 0 Ee
0 400
E 0
0
ô x
0
200
pH 5,5
0 400 0
pH 6,0
S00
v
l
pH 5,5
600
E
ô
700
0
pH 6,0
500 0 200
0
0
100
t00
pH 6,S
~
50 IO-Z(M'~) DCL X PYO. 4 . (LBFr~
0~ S ~Q~ pH 8,0
100
2
CaP " X10'3(M't)
RECIPROCAL PLOT OF INITIAL VSLOQIY AS A FUNCTION OF DI AT VARIOUB pIi's. CBS+ WA3 YSPr AT A CONÔTANT LBVtß. OF (DCL) CONCENIRATR)N, AT VARIOUS pIi'&
(DCL) CONCENTRATION,
I mM.
Cal+ was kept at a constant Level of 1 mM .
(RIOSI'~ PLOT OF RSC~ROCAL IIdIIAL Vtü." OCrrY AT V~nr~nrn Cgs+ OONC~irRATIONS.
pH was cban~ as indicated whiledicaproyllecithin concentrationwas held ounetaat at o-2 mM. Data obtained at 30°C. unchanged. However, the Michaelis constant for long chain lecithins is nearly an order of magnitude lower than that observed for the short chain homologues, suggesting a more efficient binding of long chain phospholipids to the enzyme . This effect is also reflected in the kq lKy value, which is correspondingly higher by an order of magnitude for the lecithins containing long chain fatty acid esters . The k~/Kb vs pH profile is usually a measure of ionization in the free, uncomplexed enzyme, or of the free substrate (DntoN and WESS, 1958). However, since lecithins are iso-electric over a wide pH range (pH 3"5-10) (BANaxAi~, 1963), the pK-values of 6"7 and 9"3 calculated from the log k~lKb vs pH plot must refer to groups which are part of the enzyme structure and which are free to ionize in the uncomplexed enzyme. On the other hand, the pH dependence of k~ provides the dissociation constants of groups active in catalysis (Dixox and WEBS, 1958). These groups exhibit pK-values of 6"75 and 9" 1. The near identity of the pK's derived from the kit and k~/1~Cy profiles suggests that 1~ 1 =
pH effecb on B. Gabonica 1?hospholipase As
83
0,24
a~ O,is
+1 P~ i
,= 0,12
0,08
0,04
0 -0`02 260
280
~(nm)
300
320
PiC3. S . ULTRA-VIOIE'T DIPF6aßPiCB BPSCrRA AT 1S°C OF PFIOBPHOLiPABS A : IN TRS PABSENCE of 10 - 'M C`,a'* AxD vAxYnwa pH.
- - - " pH6"3, """"""" pH6" 8,-~-~pH7"4,-x-x-x-x-x pg g.p~ --- pH 8 " 6 The protein solution contained 0"83 m8 enzyme/ml in 0" O1M HF1ES -F 0" 15M KCI. pH adjustments were made with 1"0 N HCl or 1 "0 N NaOH where neoeseary. The r~erenee cell contained enzyme solution at pH S "0. pH5"0,------pHS "8,
Ka ,r and 1~a
= 1 ~,s. This conclusion is supported by the fact that plots of 1/v vs 1/lecithin at various fixed proton concentrations intersected at a common point on the 1/substrata axis (SEGEL, 1975). Such a situation predicts that the Michaelis constant will not be affected by pH, which was indeed found in the kinetic studies. Both the acidic and basic groups must therefore be active in the breakdown of the enzyme-substrate complex (L.AmLER, 1958).
Evidently the chelation of Cat+ is governed by a groups) showing a pK--value of 6"4 (Fig. 2). This value as well as the lower of the two values derived from k~ and k~,jKb profiles (ca. 6 " 75) fall within the ionization range of the imidazole nucleus (SEGEL, 1975), while the group showing a pKvalue of ca. 9"2 may be associated with the phenolic hydroxyl of tyrosine or with amino lysine side chains (SEGEt ", 1975). Another group in the enzyme which could comply with the requirement for a pK in the pH range C~7, is a carboxyl group. TArrnoxD and TAGGART (1961) for instance, showed that a hydrophobic environment in ß-lactoglobulin causes the displacement of the pK value of a carboxyl group in
CORNSLI3 C. VILJOBN and DAWIE P. HOTES
4,0
9,0
6,0
7,0
8,0
9,0
10,0
DH FIO. 6. VARIATION OF AH80RHANCS DIFFERBNCS AT 293 IIm A3 A FUNCriON OF PSRATURB. Data were taken from Fig. 5. 0-0-15 °C; -nom 20°C ; --p-~ 25°C ; -v-v 30°C
pH
AND TFra-
Inset: Arrhenius plot of p% vs reciprocal absohrte temperature. To determine Ham , data were ßtted to equation 4. TASLS 2. pS VALUFS OF PAOBPHOLiPA88 As DSaVIi~ FROM Fta. 6 AS A FUN(,TION OF TBMPSRATUAB
Temperature (°Ci 15 20 25 30
Apparent p% values at the indicated temperature PK
S" 62 5"64 S" 66 3"68
pKs
6" 95 6" 86 6"75 6" 6?
pg~
9.35 9"22 9"15 9"OS
excess of one pH unit (i.e. from ca. 5"0 to 7 "4). In order to make more definite assignments of the groups involved in the catalytic process of phospholipase Az, determination of the enthalpies of ionization of the concerned groups was therefore attempted. This was made possible by the variation of the spectral properties of B. gabonica phospholipase Az with pH and temperature. An agreement was observed for pK values determined kinetically and those estimated from spectral data, pKal corresponding to pKz and pKa ,z to pK3. The ionizing .groups regulating the kinetic activity of B. gabonica phospholipase Az thus most likely also contribute to the spectral properties of the enzyme . The groups of pK 6"8 and 9"2 exhibit
pH e8ects on B. Gobonica PhoaphoHpeaa A,
8S
oH,,o of 7"1 Kcal/mok and 6"1 Kcal/mole respectively. A value of 7 " 1 Kcal/moie is within the range (6"8-7"3 Kcal/mok) characteristic of the heat of ionization of histidine residues (SECiBL, 197, while a ~H~ of 6"1 Kcal/mok is close to the value of 6 Kcal/mole reported by Coxi~ and Enser.c. (1943) for the tyrosine side chain. The pK of 6"4 (from kinetic data) pertaining to the group involved in Caz+ binding, is somewhat higher than pKl derived from the spectral investigations (pKi = cx. S"6~. Bearing in mind the similarity of the other pK results and limitations of the methods, pKi, and pKl are probably associated with the same group. A ~H~ of - 1"6 Kcal/mole was calculated for this group, which is characteristic of carboxyl groups (Comet and EDSAi.L, 1943). It is accepted that independence of the Michaelis constant of pH and the simultaneous variation of maximum velocity with pH, can only occur if the Michaelis constant equals the equilibrium constant for the reaction between enzyme and substrate (Coixrsa-BOWDBN, 197 . Proper protonation is therefore necessary for the catalytic step, but is of no significance in the binding step for substrate (COttI1I$H-Bovmax, 197 . In his evaluation of pH on the kinetic and spectral properties of C. adamanteus phospholipase Az, Wt3t rs (1974x) observed uacompetitive inhibition of the proton when either Caz+ or lecithin was used as variable substrates. This was ascribed to an effect of the proton on the interconversion of the central complexes and/or release of the first product (cf. Table 1 and Fig. 2, of Ws ts, 1974x) . 1~Cy was therefore found to be pH dependent, while k~lKb and K~, were shown to be unaffected by pH. However, the present study (Fig. 4) shows that the proton is a non-competitive inhibitor vs either Caz+ or lecithin as the variable substrate. A similar explanation can therefore not be offered for the inhibitory conduct of the proton . To interpret the non-competitive patterns in terms of an ordered bi-ter mechanism, at least protonation of frce enzyme also must be taken into consideration, apart from the effect the proton may have on the interconversion of the central complexes. X-ray crystallographic analysis of porcine pancreatic pre-phospholipase Az enabled Dxarrrx et al. (197 to assign functions to the side chains of Asp-56, His-55 and Tyr-35 . According to this model Asp-56 may act as nuckophile with His-55 stabilizing the putative tetrahedral intermediate formed after nucleophilic attack by Asp-56 on the carbonyl carbon of the substrate ester bond. Tyr-35 may then function as proton donor to the alkoxy-0 on C-2 which is created by hydrolysis of the ester bond. The results presented here are in agreement with the presence of tyrosine, histidine and a carboxylic acid in the active site of B. gabonica phospholipase Az. However the fact that histidine seems to be the residue responsible for the pK value of 6"75, observed in the ka,/pH plot (which provides information on groups active in catalysis), suggests that this residue, rather than the carboxylic acid side chain acts as nucleophile. Such a result is also in accord with the postulated base-catalysed mechanism of action wherein a nucleophilic attack on the ester bond by an amine is relayed through a water molecule (Sia~x and Moosex, 1975 ; Ws~s, 1974b) . The histidine residue involved, is probably His-45 of B. gxbnnica phospholipase Az. Not only is this residue conserved in all the phospholipases Az sequenced to date (Vu to$ty et al., 1977), but modification of histidine in position 45 has been shown to inactivate the enzyme (VILJOEN et al., 1977). The bromophenylacylated enzyme, although inactive, is still able to bind Caz+ as judged by the ability of this metal to induce a difference spectrum is the modified enzyme. The difference spectrum (not shown) at pH 8"0, is characterized by a large trough with a minimum at 279 nm while the difference peak observed at 242 nm (see Fig. ~, is almost abolished. In addition a positive band centering at 318 320 nm is observed, which may be ascribed to the perturbation of the newly added chromo-
86
COR.NELIS C. VII.JOEN and DAWIE P. ROTES
phore when Caz+ adds to the enzyme. The retained ability of phenacylated phospholipase A tö bind Çaz+, was. also noticed by Rolu~TS et al. (1977a) for Naja raja naja phospholipase Az and by HALP1~tT et al. (1976) for notexin, the toxic phospholipase Az isolated from Notechis scutatis scutatis . These results suggest that a residues) other than histidine is responsible for the binding of Caz+ . It is believed that Caz+ is liganded in B. gabonfca phospholipase Az by a carboxylate group having a pK value of ca. 6" 0. This value is in agreement_with the pK of 5"9 reported by ROBi~tTS et al. (1.977b) for the binding of Caz+ to the .N. naja naja enzyme . The tyrgsine ionizing at pH 9-2, the deprotonation whereof leads to inactive enzyme, obviously is a candidate for fulfilling the role of proton donor. This function and that of the carboxylate group may be provided by the invariant Tyr-35 and Asp-56 (in the Gaboon adder enzyme Tyr=2$ arYd Asp-46 ; Bo'rFS and Vu.JOSN, 1974b). However, such a prediction should be verified by specific chemical modification studies. Acknowledgements-The suthois.acknowledge the expert technical assistance of M~aTnv ùv~tvrD. REFERENCES B~vox~, A. D. (1963) Physical structure and behaviour of lipids and lipid enzymes. Adv. Lipid Res. l, 65 . Hones, D. P. and VIIJOHN, C. C. (1974x) PuriBcstIon of phospholipaso A from Bitis gabonica venom. Toxtcon 12, 611. Bores, D. P. and Vn .rosrr, C. C. (1974b) Bids gabonica venom. The amino acid sequence of phospholipase A.1. biol. Chem . 249, 3827. Cua~t~, W. W. (1963) The kinetics of ~zyme-catalyzed reactions with two or more substrates or products L Nomenclature and rate equations. Biochim. biophys. Acts 67, 104. Germ, W. W. (1967) Tho atatistIcal analysis of enzyme kinetic data. Adv. Enzymol. 29, 1. G~urro, W. W. (1970) Steady state kinetics. In : The Fiezymes, Vol. 2, p. 1, (HoYex, P. D., Ed). New York : Aczu~ic Press. Come, E. J. and E~"?~, J. T. (1943) Proteins, Amino Acids and Peptides, p. 444. New York : Reinhold Publishing. CoRrusa-Bownerr, A. (1976) Principles of Enzyme Küutics, p: 101. London : Buttorworths . Dixox, M. aad WHes, E. C. (1958) Enzymes, let Edn., p. 120 London : Longmans oreen. Dxe?rra, J., Errz«o, C. M., K~c, K. H. and Vests, J. C. A. (1976) Structure of porcine pancreatic prephospholipase As. Nature, Lond. 264, 373. HALPERT, J., E~s~e, D. and K~ox, E. (1976) . The role of phospholi~se activity in the action of a presynaptic neurotoxin from the venom of NotecMs scutatis scutaNs (Australian Tiger Snake) . FEBS Lett . 61, 72. Lr~tas, K. J. (1968) The Chemical %inetics of Enzyme Action, p. 117. London : Oxford University Press. Revsma., F. M. and Crlßwrm, W. W. (1977) Determination of the rats~limiting steps and chemical mechanisms of fivctokinase by isotope axchaage, isotope partitioning and pH studies. Biochemistry 16, 2176. Ro~~rs, M. F., Ds®ts, R. A., MrNC~, T. C. and DeNtus, E. A. (1977x) Chemical modification of the histidine.residue in phospholipaso A~ (Ngja n~a ngja).1. blot. Chem. 252, 2405 . Ro~zs, M. F., Deems, R. A, and Dexr~, E. A. (1977b) Spectral perturbations of the histidine and tryptophan in cobra venom phospholipase Ax upon metal ion and mixed micelle binding. J. btol. Chem . 252, 6011 . Roxorr, O. A. and Scmr+t~ownz, M. (1961) Studica of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A. Archs Biodiem. Biophys. 94, 364. 3Has., I. H. (1975) Enzyme Emetics, p. 884. New York : John Wiley and Sons. Sioux, D. S. and Moose, C3. (1975) Chemical studies of enzyme action sites. A. Rev. Biochem . 44, 889. Tetu+oxn, C. and T~aowxz, V. C~ . (1961) Ionization-linked changes in protein conformation-II. The N -s R transition is ß-lactoglobulin, l. Am . them. Soe. 83, 1634. Vuaoex, C. C., Sceusoar, J. C. and Hares, D. P. (1974) Bitis gabonica venom. A kinetic analysis of the hydrolysis by phospholipese Az of 1,7rdipalmitoyl~n-glycero-3-phosphorylci~oline . Biochtm. btophys. Acts 360,156. Vnaoex, C. C., Bores, D. P., and SCHAHORT J. .C . (1975) Spectral properties of Bitis gabontca venom phospholipase A, is the presence of divalent metal ion, substrate and hydrolysis products. Toxicon 13, 343. VII.roeav, C. C., Ve~ax, L. and Bores, D. P. (1976) An essential tryptophan in the active site of phospholipase Az from the venom of Bitis gabonica. BiocMm. blophys . Acts 438, 424.
pH effects on B. Gaborelca Phoapholipasa As
87
VII.rosrr, C. C., VISSER, L . and Harea, D. P. (1977) Histidine aad (ysina residues and tha activity of phospholipase As from the v~om of BJt1J gabonica . Biochim. biophya . .lcta 483,107, Wrffas, M. A. (1972) A kinetic study of the phospholipase As (GYotales adansmrtera) catalyzed hydrolysis of 1,2-dibutyryl-sn-glyo~o-3-phosphorykholina . BiochemLstry 11,1030. W~.ts, M. A. (1973) Effects of chemical modification on the activity of Crotales adamantees phospholipase As. Evidonoo for an essential amino soup. Btoclunilstry 12, 1086. Wsua, M. A. (1974x) Effort of pH on the kinetic aad spaxral properties of Crotales admnmrtees phospholipase As in Hs0 aad DsO . Blochardstry 13, 2265 . W as, M. A. (19746) A phospholipaso As model systems. Calcium eahancem~t of the amine~atalyred methanolysis of phosphatidykholiae . Blochanistry 13, 2258.
, vo~ . r7,yp~ rt-8~r. Pmp Ltd. 1979.
oo~i-oioil79pioi-oo~nsozoo~o
Printed la t3reet Hrltain.
INFLUENCE OF pH ON THE KINETIC AND SPECTRAL PROPERTIES OF PHOSPHOLIPASE A2 FROM BITIS GABONICA (GABOON ADDER) SNAKE VENOM CORNELIS C. VIIJOSN
and Dewy P. Borns
Molecular Biochemistry Division, National Q>ennical Research Laboratory, Council for Sci~tific and Industrial Iteeearch, P.O. Hox 395, Pretoria, 0001, Republic of South Africa (dcceptedjor publlaatlon 28 .lpril 1978)
C. C. Vii.Ta~v and D. P. Ho~res . I~ueaoe of pH oa the kinetic and spectral properties of phospholipase A= from Blur gabortka (Ciaboon adder) snake venom. Toxlcon 17, 77-87, 1979.-The i~uenoe of pH on the kinetic parameteas ~Cae and ka,/%b, and the spectral properties of BitiCr gabonica phospholipase A~, showed that the activity of the enzyme is controlled by groups of p% values 6"8 and 9"2. These groups were found to exhibit enthalpies of ionization (nH~ of 7"1 Kcal/mole and 6"1 Keàl/mole respectively which are eharactcristic of nH~ of histidine sad tyrosine residues. Whilo the dissociation constant (%~ for reaction between enzyme and Cap* was affected by pH, the Michaelis constant (%~ of the enzyme for thophoapholipid substrate was found to ba uninfluenced in the pH range tented i.e. pH S"S-A"0. The binding of Ca3+ to tl~ enzyme is inhibited by protonation of a group of p% ca. 6-0. The oH~ of - 1 "6 Kcal/mole cakarlated for tl»s group is is the rango of the values found for carboiryl groups. The various functions of nucleophi~e, proton donor and Ca3* binding site may be aselgried to the side chains of atructuraliy invariant residues which for the B. gabo,dca plioapliolipase As are located at His-45, Tyt-25 and Asp~6 respectively" Non~ompetltive inhibition of the H+ was obe~ved with respect to both Ca3* and lecithin wlüch probabiy involves an effect of theproton oa feeo ~and theinterconveraioa of theoeatraloomplexee. iIVTRODUCTiON
studies on snake venom phospholipase Az (EC 3.1.1 .4) have shown that the enzyme follows a kinetic mechanism of the ordered bi-ten type (W~.rs, 1972 ; VIIJOSN et al., 1974). Also group specific chemical modification inferred certain residues as funotionally important, either in substrate binding, or the catalytic process itself (W1u.1s, 1973 ; Vu row et x1.,1977) . Wells, from an evaluation ofthe pH dependence of the reaction, concluded that a group with a pK value of 7"6 was involved in the catalytic step of the reaction catalysed by Crotalus adamanteus phospholipase Az ~ "" B[.TS, 1972 ; 1973 ; 1974x). Previous studies on the influence of pH on the rate of the hydrolytic reaction catalysed by Bltis gabonlca phospholipase Az was in accord with Wells' finding (VII.TOSrr et al., 1974). A more detailed investigation of the variation of the kinetic parameters of this enzyme with pH however, did not confirm the previous results. The findings of this study are reported here. PRBVIOUS
MoteHaLr
MATR.RTAi c AND METHODS
Dicaproyl-t-ao-ledthin wan obtained from Applied Science Laboratories (USA) and caproic acid wan a product of Fsstman Organic Chemicals (USA). h?hospholipase As purified from the venom of B . gabonica as described (Hares end Vn row, 1974x) was used as enzyme source . Fnzymo ooaceatration was determined from absorbanee values and e= -2"52 x 10 " M- ~cm_r (V>t.roert, 1974, Ph.D. Thesis, Read Afrikaans University, Johannesburg, Republic of South Afrka).
78
CORNELIS C. VIIJOEN aad DAV1~ P. BOTES
Kfxetlc detamfnxtion Activity ofphsopholipase As was mesa~ued by the acIdimetric sassy described previously (VII.AOBN et ol., 1974) sad modified as follows. The reaction was carried out is a Radiometer TTA31 titration assembly connected via a C3I{2321C combined glass sad reietence electrode to a Radiometer 26 ~rpanded scale pH meter. Liberation of five fatty acid was monitored continuously on a Bechmaa Tea Inch Pot~tiometric Recorder. Recorder response was standardised with 10'sM caproic acid. Full~cale sensitivity of 0"2 pH units sad a Chart epeod of 1-10 in/min wale used. Moaaureo>~ta were obtained in theamostated reaction mixtures of 2 ml total vofime under a constant stroam of nitrogen. Reaction mixtures contained 0"15M KCl and CaQs and dkaproyllecithin at the apatifled concentrations. All concentrations of substrate used is the exPerlmeats reported here, were below the critical micelle oonoentration of 0"01 M (ROHOLT and Scn~owrrz, 1%1). A minimum enzyme molecular weight of 13347 (Bo~aa sad Vn wo~r, 1974x) was used to eapr~ess reaction rates in turnovea numbeaa . Malysis ojkixetlc and spectral data The nom~clature of this paper is that of C~Nn (1963) . Initial velocity data wem first analysed graphically in double reciprocal plots of initial velodty/substrate concentration to check linearity and pattern of the plots. Data conforming to equilibrium ordered initial velocity were ßtted to equation 1. YulB Kb.! -I- .!B -Igtagb
v ~
Y
( C ~ ~`1 -f- H/g~ ~- Ks/H)
~
Y
i2) (3)
- ~ \ 1 -I- H/g~ /
In equation 1, v, V,, Z~ sad Ky have the meanings described previously, v and V, reprneeating the observed and maximum velocities, respectively while K~ is the dissociation constant for substrate A sad S~, the Michselis constant for substrate B (VII.~osrr et al., 1974). pH profiles wero fitted either to equation 2 or equation 3 whero K, and 8s represent dissociation constants of groups that moat be deprotonated or protonaied respectively, for activity, H is the hydrogen-ion oonoeatration, and C is the value of Y, the pH dependent ]vnetic parameter, at the optimum state ofprotonation. Arrhenius plots of (data from spectroscopy studies) were fitted to equation 4 (Rwss~. sad C~xn, 1977), where Y equals pg, T the
pSlT
absolute tempeaature, a -2~,3H03R and b i, a constant. Experimental data were fitted to equations 1-4 by the least aquat+es method deecnôed by CtEUr~ (1967) . The points in the plots indicate exPerimontal data, while the curves wero calculated from ßta to the equations above. SP~~~PY Differraoe spectroscopy were carried out as described (Vn ronrr et x1.,1975) is e Varias Techtron Model 633 double beam spectropimtometer coupled to a Hitachi Model QPD73 recorder, and thermoatated by menas o~f a Lauds Ultra Kryomat Model TK-30-D.
RESULTS The kinetic parameters for the reaction between phoapholipase A2 and dicaproyllecithin were obtained from initial velocity studies carried out as a function of either Cap+ or dicaproyllectithin concentration at changing fixed concentrations of the other substrate . Figure 1 illustrates the results of such an experiment at pH 8"0 and 30°C. Using Caz+ as the variable substrate, a family of straight lines were obtained intersecting to the left of the vertical axis, while a family of plots intersecting on the vertical axis were observed when dicaproyllecithin was used as variable substrate at various fixed Caz+ concentrations. Values for Kr Kb and Vr were calculated from a fit of the data to equation 1. The influence of pH on these parameters wen investigated by performing identical experiments in the pH range 5"5-9"0.
pH ~ects on
B. GaboniQO
2S
60
79
Phwpholipese As
TS
100
Dc~ X~0-2(M~I)
FIO. 1. (A) REC~ROCAL PLOT OF INITIAL VELOCITY Aô A FUNCIiON OP Cas+ WITH DICAPROYIr LSQTHIN (DCL) AS THE st)Ti~ATS.
Reaction was initiated with 14 I+g of an ~zyme solution in distr7led water. The numbers on the right indicato the fixed levels of dicaproyllecithin. Data ware collected at pH 8"0 and 30°C . (B) Reciprocal plot of initial velocity as a function of dicaproyllecithin (DCL) oonoentratioa at various levels of Cas+ . Data were obtained at pH 8"0 and 30°C.
Table 1 compares the kinetic constants obtained for the catalysis by phospholipase Az of the hydrolysis of dipalmitoyllecithin with that of the substrate of the present study. It is evident that only the Michaelis constant and therefore the parameter k~ JKb, were influenced by using dicaproyllecithin as substrate for the enzyme . TAeLE 1. COI~ARDON OF TH8 LII~IIC PARAM611~ OF B. 8abvniao Paa~HpL>PASB AT pH 8"0 AND 2O°C OF DIPALAnT+0YI18CTIId AND DI
$Ubstrate
S;~
As
FoR THS xYDROLYSis
KlnOelC paramDt~ Sb ~
kat
~%b
(min - ') (min-' M-') Dicaproyllxithin 4"6 x 10 - 4 S"6 x 10- ~ 4"8 x lOs 8"77 x 10= Dipabnitoylkcithin' 8 x 10'~ 3"2 x 10- ~ 6"Ol x lOs 1"88 x 10~ 'Values reported previously (VILJOBN et 4.,1974). Ia this publication ~ead%b was incorrectly given as 830 M' l.s'' while it should read 8"3 x 10~ M' l.a'~. The values of k~ , and ~.d%b wero based on a mol . wt of 30,000 and aro for reasons of comparison corrected to the mol. wt of 13347 used in tho present report. t%, represents the dissociation constant for substrate A, %b the Michaolis constant for substrate B .k~,t the catalytic rate constant, obtained by divIdiag V, (maximum velocity) by total enzyme conc~tration, whsle Jfc~,~/%b is the apparent first order rate constant for the reaction of enzyme with substrate at low ooncemtrations of substrate. (M)
COItNELL4 G VIUOEN and DAWIS P. ROTES
3,3
a ô 3,2
0 0
0
0
3,1 0
3,2
0 G
0
0 0
0
2,8
Y
2,4
2,0 5,0
Ei,O
7,0 pH
8,0
9,0
Fw. 2. (Top) Ixnvswce oa pH ox z~ IVlzcae~ corrsr~xr Kb . Data were collexted from experiments similar to those described in Fig. l, at the indicated pH's and a bemparature of 30°C. (Bo~ri~) pH VARTATION OF Kh, nrrreo ~ro ~trszmx 3, pK - 6"4.
Figure 2 shows that pKb is pH independent and a mean value of 5"9 x 10-+M was calculated for this parameter. However, the dissociation constant (K~ for the binding of Caz+ to the enzyme changed with pH (Fig. 2). The experimental data were fitted to equation 3, giving a pK of 6"4. The effect of pH on k~ is depicted in Fig. 3, showing the lxll-shaped characteristics of the profile. The data were therefore fitted to equation 2, resulting in pKvalues of p~,l = 6"75 and p1~Ca,z = 9"1. A similar curve was obtained for the variation of log kq~Kb with pH (Fig. 3). The pK's from a fit of the experimental values to equation 2, were estimated as ply, = 6"7 and p1~s = 9" 3. Values of the kinetic parameter at the optimum pH were calculated from the various fits as k~~ = 8 " 57 x 10~ min -', kajKb = 1"35 x 106 mini M- 1 and K~ = 7"24 x 10- 4M. Examination of the effect of pH on initial velocity, data, demonstrated that the proton acts as a noncompetitive inhibitor with respect to both Caz+ and dicaproyllecithin (Fig. 4). In both instances the family of straight lines obtained when reciprocal initial velocity was plotted against reciprocal substrate at various pH's, intersected to the left of the ordinate, but only in the case of dicaproyllocithin as variable substrate did the lines intersect on the 1/Substrato-axis . It has already been found that binding of Cat+ to the enzyme causes perturbation of tryptophan and tyrosine residues (Vu.~o~r et al., 197 . Figure 5 illustrates this phenomenon at various pH's. From the pH dependence of the change in absorbance at 293 nm at least 3 transition zones could be discerned, centering at about pH 5"6, 6"8 and 9"2 (Fig. 6). The variation of the apparent pK values with temperature is set out in Table 2. These
pH effexts on ~. Gabontex+ Phospholipate As
Sl
6,0
4,8
2,8 ô
2,O
~'~,o
s,o
~,o
e,o
s,o
pH Fix. 3. (Tor) EFFHCr of pH oN lo$ ~OUIKn . The line represents a flt to equation 2, pge , - 6~7 and pKa, - 9"3: pKa + and pl~z rda to the dissociation of groups in free, uncompleAed eazyme . ~HOTPOIN) pH VARrATiON OF l0$ Jkat" Data ware fitted to equation 2, pga+ - fr75 and pSa,s - 9~1 ; pKe,+ and pge,~ mist to the dissociation of groups in the mzyme~ubstrate complex. The parameters lc~ aad k~/Kt, were obtained from initial velocity experiments such as described is Fig. 1, at the indicated pH's . Experiments were carried out at 30°C.
values were fitted to equation 4 to calculate the heat of ionization of groups associated with the various pK values . Enthalpies of ionization of - 1 "6 Kcal/mole, 7" 1 Kcal/mole and 6" 1 Kcal/mole were thus estimated for the groups of pK,, pKs aad pK3 respectively . DISCUSSION
The initial velocity patterns observed in this study showed the diagnostic features for an equilibrium ordered addition of substrates, wherein the addition of Cap+ to the enzyme is followed by addition of lecithin, while the binding of metal to the enzyme is at thermodynamic equilibrium (Cr.rrr ."t~m, 1970). These results confirm those previously obtained using dipahnitoyllecithin as substrate (VtuosN et al., 1974). As can be gathered from Table 1, the long chain lecithins constitute better substrates for the enzyme than the phospholipids containing short chain fatty acid esters . The influence of substrate is not seen on the catalytic constant of the enzyme, since this value is, within experimental error,
CORNELIS C. VILTOEN and DAV1+18 P. HOTPS
82
700
I
0
ô E c É
x
0 600
500
500 0 Ee
0 400
E 0
0
ô x
0
200
pH 5,5
0 400 0
pH 6,0
S00
v
l
pH 5,5
600
E
ô
700
0
pH 6,0
500 0 200
0
0
100
t00
pH 6,S
~
50 IO-Z(M'~) DCL X PYO. 4 . (LBFr~
0~ S ~Q~ pH 8,0
100
2
CaP " X10'3(M't)
RECIPROCAL PLOT OF INITIAL VSLOQIY AS A FUNCTION OF DI AT VARIOUB pIi's. CBS+ WA3 YSPr AT A CONÔTANT LBVtß. OF (DCL) CONCENIRATR)N, AT VARIOUS pIi'&
(DCL) CONCENTRATION,
I mM.
Cal+ was kept at a constant Level of 1 mM .
(RIOSI'~ PLOT OF RSC~ROCAL IIdIIAL Vtü." OCrrY AT V~nr~nrn Cgs+ OONC~irRATIONS.
pH was cban~ as indicated whiledicaproyllecithin concentrationwas held ounetaat at o-2 mM. Data obtained at 30°C. unchanged. However, the Michaelis constant for long chain lecithins is nearly an order of magnitude lower than that observed for the short chain homologues, suggesting a more efficient binding of long chain phospholipids to the enzyme . This effect is also reflected in the kq lKy value, which is correspondingly higher by an order of magnitude for the lecithins containing long chain fatty acid esters . The k~/Kb vs pH profile is usually a measure of ionization in the free, uncomplexed enzyme, or of the free substrate (DntoN and WESS, 1958). However, since lecithins are iso-electric over a wide pH range (pH 3"5-10) (BANaxAi~, 1963), the pK-values of 6"7 and 9"3 calculated from the log k~lKb vs pH plot must refer to groups which are part of the enzyme structure and which are free to ionize in the uncomplexed enzyme. On the other hand, the pH dependence of k~ provides the dissociation constants of groups active in catalysis (Dixox and WEBS, 1958). These groups exhibit pK-values of 6"75 and 9" 1. The near identity of the pK's derived from the kit and k~/1~Cy profiles suggests that 1~ 1 =
pH effecb on B. Gabonica 1?hospholipase As
83
0,24
a~ O,is
+1 P~ i
,= 0,12
0,08
0,04
0 -0`02 260
280
~(nm)
300
320
PiC3. S . ULTRA-VIOIE'T DIPF6aßPiCB BPSCrRA AT 1S°C OF PFIOBPHOLiPABS A : IN TRS PABSENCE of 10 - 'M C`,a'* AxD vAxYnwa pH.
- - - " pH6"3, """"""" pH6" 8,-~-~pH7"4,-x-x-x-x-x pg g.p~ --- pH 8 " 6 The protein solution contained 0"83 m8 enzyme/ml in 0" O1M HF1ES -F 0" 15M KCI. pH adjustments were made with 1"0 N HCl or 1 "0 N NaOH where neoeseary. The r~erenee cell contained enzyme solution at pH S "0. pH5"0,------pHS "8,
Ka ,r and 1~a
= 1 ~,s. This conclusion is supported by the fact that plots of 1/v vs 1/lecithin at various fixed proton concentrations intersected at a common point on the 1/substrata axis (SEGEL, 1975). Such a situation predicts that the Michaelis constant will not be affected by pH, which was indeed found in the kinetic studies. Both the acidic and basic groups must therefore be active in the breakdown of the enzyme-substrate complex (L.AmLER, 1958).
Evidently the chelation of Cat+ is governed by a groups) showing a pK--value of 6"4 (Fig. 2). This value as well as the lower of the two values derived from k~ and k~,jKb profiles (ca. 6 " 75) fall within the ionization range of the imidazole nucleus (SEGEL, 1975), while the group showing a pKvalue of ca. 9"2 may be associated with the phenolic hydroxyl of tyrosine or with amino lysine side chains (SEGEt ", 1975). Another group in the enzyme which could comply with the requirement for a pK in the pH range C~7, is a carboxyl group. TArrnoxD and TAGGART (1961) for instance, showed that a hydrophobic environment in ß-lactoglobulin causes the displacement of the pK value of a carboxyl group in
CORNSLI3 C. VILJOBN and DAWIE P. HOTES
4,0
9,0
6,0
7,0
8,0
9,0
10,0
DH FIO. 6. VARIATION OF AH80RHANCS DIFFERBNCS AT 293 IIm A3 A FUNCriON OF PSRATURB. Data were taken from Fig. 5. 0-0-15 °C; -nom 20°C ; --p-~ 25°C ; -v-v 30°C
pH
AND TFra-
Inset: Arrhenius plot of p% vs reciprocal absohrte temperature. To determine Ham , data were ßtted to equation 4. TASLS 2. pS VALUFS OF PAOBPHOLiPA88 As DSaVIi~ FROM Fta. 6 AS A FUN(,TION OF TBMPSRATUAB
Temperature (°Ci 15 20 25 30
Apparent p% values at the indicated temperature PK
S" 62 5"64 S" 66 3"68
pKs
6" 95 6" 86 6"75 6" 6?
pg~
9.35 9"22 9"15 9"OS
excess of one pH unit (i.e. from ca. 5"0 to 7 "4). In order to make more definite assignments of the groups involved in the catalytic process of phospholipase Az, determination of the enthalpies of ionization of the concerned groups was therefore attempted. This was made possible by the variation of the spectral properties of B. gabonica phospholipase Az with pH and temperature. An agreement was observed for pK values determined kinetically and those estimated from spectral data, pKal corresponding to pKz and pKa ,z to pK3. The ionizing .groups regulating the kinetic activity of B. gabonica phospholipase Az thus most likely also contribute to the spectral properties of the enzyme . The groups of pK 6"8 and 9"2 exhibit
pH e8ects on B. Gobonica PhoaphoHpeaa A,
8S
oH,,o of 7"1 Kcal/mok and 6"1 Kcal/mole respectively. A value of 7 " 1 Kcal/moie is within the range (6"8-7"3 Kcal/mok) characteristic of the heat of ionization of histidine residues (SECiBL, 197, while a ~H~ of 6"1 Kcal/mok is close to the value of 6 Kcal/mole reported by Coxi~ and Enser.c. (1943) for the tyrosine side chain. The pK of 6"4 (from kinetic data) pertaining to the group involved in Caz+ binding, is somewhat higher than pKl derived from the spectral investigations (pKi = cx. S"6~. Bearing in mind the similarity of the other pK results and limitations of the methods, pKi, and pKl are probably associated with the same group. A ~H~ of - 1"6 Kcal/mole was calculated for this group, which is characteristic of carboxyl groups (Comet and EDSAi.L, 1943). It is accepted that independence of the Michaelis constant of pH and the simultaneous variation of maximum velocity with pH, can only occur if the Michaelis constant equals the equilibrium constant for the reaction between enzyme and substrate (Coixrsa-BOWDBN, 197 . Proper protonation is therefore necessary for the catalytic step, but is of no significance in the binding step for substrate (COttI1I$H-Bovmax, 197 . In his evaluation of pH on the kinetic and spectral properties of C. adamanteus phospholipase Az, Wt3t rs (1974x) observed uacompetitive inhibition of the proton when either Caz+ or lecithin was used as variable substrates. This was ascribed to an effect of the proton on the interconversion of the central complexes and/or release of the first product (cf. Table 1 and Fig. 2, of Ws ts, 1974x) . 1~Cy was therefore found to be pH dependent, while k~lKb and K~, were shown to be unaffected by pH. However, the present study (Fig. 4) shows that the proton is a non-competitive inhibitor vs either Caz+ or lecithin as the variable substrate. A similar explanation can therefore not be offered for the inhibitory conduct of the proton . To interpret the non-competitive patterns in terms of an ordered bi-ter mechanism, at least protonation of frce enzyme also must be taken into consideration, apart from the effect the proton may have on the interconversion of the central complexes. X-ray crystallographic analysis of porcine pancreatic pre-phospholipase Az enabled Dxarrrx et al. (197 to assign functions to the side chains of Asp-56, His-55 and Tyr-35 . According to this model Asp-56 may act as nuckophile with His-55 stabilizing the putative tetrahedral intermediate formed after nucleophilic attack by Asp-56 on the carbonyl carbon of the substrate ester bond. Tyr-35 may then function as proton donor to the alkoxy-0 on C-2 which is created by hydrolysis of the ester bond. The results presented here are in agreement with the presence of tyrosine, histidine and a carboxylic acid in the active site of B. gabonica phospholipase Az. However the fact that histidine seems to be the residue responsible for the pK value of 6"75, observed in the ka,/pH plot (which provides information on groups active in catalysis), suggests that this residue, rather than the carboxylic acid side chain acts as nucleophile. Such a result is also in accord with the postulated base-catalysed mechanism of action wherein a nucleophilic attack on the ester bond by an amine is relayed through a water molecule (Sia~x and Moosex, 1975 ; Ws~s, 1974b) . The histidine residue involved, is probably His-45 of B. gxbnnica phospholipase Az. Not only is this residue conserved in all the phospholipases Az sequenced to date (Vu to$ty et al., 1977), but modification of histidine in position 45 has been shown to inactivate the enzyme (VILJOEN et al., 1977). The bromophenylacylated enzyme, although inactive, is still able to bind Caz+ as judged by the ability of this metal to induce a difference spectrum is the modified enzyme. The difference spectrum (not shown) at pH 8"0, is characterized by a large trough with a minimum at 279 nm while the difference peak observed at 242 nm (see Fig. ~, is almost abolished. In addition a positive band centering at 318 320 nm is observed, which may be ascribed to the perturbation of the newly added chromo-
86
COR.NELIS C. VII.JOEN and DAWIE P. ROTES
phore when Caz+ adds to the enzyme. The retained ability of phenacylated phospholipase A tö bind Çaz+, was. also noticed by Rolu~TS et al. (1977a) for Naja raja naja phospholipase Az and by HALP1~tT et al. (1976) for notexin, the toxic phospholipase Az isolated from Notechis scutatis scutatis . These results suggest that a residues) other than histidine is responsible for the binding of Caz+ . It is believed that Caz+ is liganded in B. gabonfca phospholipase Az by a carboxylate group having a pK value of ca. 6" 0. This value is in agreement_with the pK of 5"9 reported by ROBi~tTS et al. (1.977b) for the binding of Caz+ to the .N. naja naja enzyme . The tyrgsine ionizing at pH 9-2, the deprotonation whereof leads to inactive enzyme, obviously is a candidate for fulfilling the role of proton donor. This function and that of the carboxylate group may be provided by the invariant Tyr-35 and Asp-56 (in the Gaboon adder enzyme Tyr=2$ arYd Asp-46 ; Bo'rFS and Vu.JOSN, 1974b). However, such a prediction should be verified by specific chemical modification studies. Acknowledgements-The suthois.acknowledge the expert technical assistance of M~aTnv ùv~tvrD. REFERENCES B~vox~, A. D. (1963) Physical structure and behaviour of lipids and lipid enzymes. Adv. Lipid Res. l, 65 . Hones, D. P. and VIIJOHN, C. C. (1974x) PuriBcstIon of phospholipaso A from Bitis gabonica venom. Toxtcon 12, 611. Bores, D. P. and Vn .rosrr, C. C. (1974b) Bids gabonica venom. The amino acid sequence of phospholipase A.1. biol. Chem . 249, 3827. Cua~t~, W. W. (1963) The kinetics of ~zyme-catalyzed reactions with two or more substrates or products L Nomenclature and rate equations. Biochim. biophys. Acts 67, 104. Germ, W. W. (1967) Tho atatistIcal analysis of enzyme kinetic data. Adv. Enzymol. 29, 1. G~urro, W. W. (1970) Steady state kinetics. In : The Fiezymes, Vol. 2, p. 1, (HoYex, P. D., Ed). New York : Aczu~ic Press. Come, E. J. and E~"?~, J. T. (1943) Proteins, Amino Acids and Peptides, p. 444. New York : Reinhold Publishing. CoRrusa-Bownerr, A. (1976) Principles of Enzyme Küutics, p: 101. London : Buttorworths . Dixox, M. aad WHes, E. C. (1958) Enzymes, let Edn., p. 120 London : Longmans oreen. Dxe?rra, J., Errz«o, C. M., K~c, K. H. and Vests, J. C. A. (1976) Structure of porcine pancreatic prephospholipase As. Nature, Lond. 264, 373. HALPERT, J., E~s~e, D. and K~ox, E. (1976) . The role of phospholi~se activity in the action of a presynaptic neurotoxin from the venom of NotecMs scutatis scutaNs (Australian Tiger Snake) . FEBS Lett . 61, 72. Lr~tas, K. J. (1968) The Chemical %inetics of Enzyme Action, p. 117. London : Oxford University Press. Revsma., F. M. and Crlßwrm, W. W. (1977) Determination of the rats~limiting steps and chemical mechanisms of fivctokinase by isotope axchaage, isotope partitioning and pH studies. Biochemistry 16, 2176. Ro~~rs, M. F., Ds®ts, R. A., MrNC~, T. C. and DeNtus, E. A. (1977x) Chemical modification of the histidine.residue in phospholipaso A~ (Ngja n~a ngja).1. blot. Chem. 252, 2405 . Ro~zs, M. F., Deems, R. A, and Dexr~, E. A. (1977b) Spectral perturbations of the histidine and tryptophan in cobra venom phospholipase Ax upon metal ion and mixed micelle binding. J. btol. Chem . 252, 6011 . Roxorr, O. A. and Scmr+t~ownz, M. (1961) Studica of the use of dihexanoyllecithin and other lecithins as substrates for phospholipase A. Archs Biodiem. Biophys. 94, 364. 3Has., I. H. (1975) Enzyme Emetics, p. 884. New York : John Wiley and Sons. Sioux, D. S. and Moose, C3. (1975) Chemical studies of enzyme action sites. A. Rev. Biochem . 44, 889. Tetu+oxn, C. and T~aowxz, V. C~ . (1961) Ionization-linked changes in protein conformation-II. The N -s R transition is ß-lactoglobulin, l. Am . them. Soe. 83, 1634. Vuaoex, C. C., Sceusoar, J. C. and Hares, D. P. (1974) Bitis gabonica venom. A kinetic analysis of the hydrolysis by phospholipese Az of 1,7rdipalmitoyl~n-glycero-3-phosphorylci~oline . Biochtm. btophys. Acts 360,156. Vnaoex, C. C., Bores, D. P., and SCHAHORT J. .C . (1975) Spectral properties of Bitis gabontca venom phospholipase A, is the presence of divalent metal ion, substrate and hydrolysis products. Toxicon 13, 343. VII.roeav, C. C., Ve~ax, L. and Bores, D. P. (1976) An essential tryptophan in the active site of phospholipase Az from the venom of Bitis gabonica. BiocMm. blophys . Acts 438, 424.
pH effects on B. Gaborelca Phoapholipasa As
87
VII.rosrr, C. C., VISSER, L . and Harea, D. P. (1977) Histidine aad (ysina residues and tha activity of phospholipase As from the v~om of BJt1J gabonica . Biochim. biophya . .lcta 483,107, Wrffas, M. A. (1972) A kinetic study of the phospholipase As (GYotales adansmrtera) catalyzed hydrolysis of 1,2-dibutyryl-sn-glyo~o-3-phosphorykholina . BiochemLstry 11,1030. W~.ts, M. A. (1973) Effects of chemical modification on the activity of Crotales adamantees phospholipase As. Evidonoo for an essential amino soup. Btoclunilstry 12, 1086. Wsua, M. A. (1974x) Effort of pH on the kinetic aad spaxral properties of Crotales admnmrtees phospholipase As in Hs0 aad DsO . Blochardstry 13, 2265 . W as, M. A. (19746) A phospholipaso As model systems. Calcium eahancem~t of the amine~atalyred methanolysis of phosphatidykholiae . Blochanistry 13, 2258.
