pH dependence of light-driven proton pumping by an archaerhodopsin from Tibet: comparison with bacteriorhodopsin

The pH-dependence of photocycle of archaerhodopsin 4 (AR4) was examined, and the underlying proton pumping mechanism investigated. AR4 is a retinal-containing membrane protein isolated from a strain of halobacteria from a Tibetan salt lake. It acts as a light-driven proton pump like bacteriorhodopsin (BR). However, AR4 exhibits an "abnormal" feature--the time sequence of proton release and uptake is reversed at neutral pH. We show here that the temporal sequence of AR4 reversed to "normal"--proton release preceding proton uptake--when the pH is increased above 8.6. We estimated the pK(a) of the proton release complex (PRC) in the M-intermediate to be approximately 8.4, much higher than 5.7 of wide-type BR. The pH-dependence of the rate constant of M-formation shows that the pK(a) of PRC in the initial state of AR4 is approximately 10.4, whereas it is 9.7 in BR. Thus in AR4, the chromophore photoisomerization and subsequent proton transport from the Schiff base to Asp-85 is coupled to a decrease in the pK(a) of PRC from 10.4 to 8.4, which is 2 pK units less than in BR (4 units). This weakened coupling accounts for the lack of early proton release at neutral pH and the reversed time sequence of proton release and uptake in AR4. Nevertheless the PRC in AR4 effectively facilitates deprotonation of primary proton acceptor and recovery of initial state at neutral pH. We found also that all pK(a)s of the key amino acid residues in AR4 were elevated compared to those of BR.     

Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d

Sac7d and Sso7d are homologous, hyperthermophile proteins with a high density of charged surface residues and potential ion pairs. To determine the relative importance of specific amino acid side-chains in defining the stability and function of these Archaeal chromatin proteins, pK(a) values were measured for the acidic residues in both proteins using (13)C NMR chemical shifts. The stability of Sso7d enabled titrations to pH 1 under low-salt conditions. Two aspartate residues in Sso7d (D16 and D35) and a single glutamate residue (G54) showed significantly perturbed pK(a) values in low salt, indicating that the observed pH-dependence of stability was primarily due to these three residues. The pH-dependence of backbone amide NMR resonances demonstrated that perturbation of all three pK(a) values was primarily the result of side-chain to backbone amide hydrogen bonds. Few of the significantly perturbed acidic pK(a) values in Sac7d and Sso7d could be attributed to primarily ion pair or electrostatic interactions. A smaller perturbation of E48 (E47 in Sac7d) was ascribed to an ion pair interaction that may be important in defining the DNA binding surface. The small number (three) of significantly altered pK(a) values was in good agreement with a linkage analysis of the temperature, pH, and salt-dependence of folding. The linkage of the ionization of two or more side-chains to protein folding led to apparent cooperativity in the pH-dependence of folding, although each group titrated independently with a Hill coefficient near unity. These results demonstrate that the acid pH-dependence of protein stability in these hyperthermophile proteins is due to independent titration of acidic residues with pK(a) values perturbed primarily by hydrogen bonding of the side-chain to the backbone. This work demonstrates the need for caution in using structural data alone to argue the importance of ion pairs in stabilizing hyperthermophile proteins.     

Golgi β-glucuronidase of androgen-stimulated mouse kidney

The equilibrium constant of the phosphoglyceromutase reaction was determined over a range of pH (5.4-7.9), in solutions of different ionic strength (0.06-0.3) and in the presence of Mg(2+), at 30 degrees C and at 20 degrees C. The values obtained (8.65-11.65) differ substantially from previously published values. The third acid dissociation constants were redetermined for 2- and 3-phosphoglycerate, and in contrast with previous reports the pK values (7.03 and 6.97 respectively at zero ionic strength) were closely similar. The Mg(2+)-binding constants were measured spectrophotometrically and the values, 286mm(-1) and 255mm(-1) for 2- and 3-phosphoglycerate at pH7 and ionic strength 0.02, were also very similar. From the relative lack of effect of temperature, pH and ionic strength it is concluded that the equilibrium constant differs from unity largely because of entropic factors. At low ionic strength, in the neutral region, the pH-dependence can be attributed to the small difference in the acid dissociation constants, but the difference in dissociation constants does not explain the pH-dependence in the acid region or at high ionic strength. Within physiological ranges of pH, Mg(2+) concentration and ionic strength there will be little variation in equilibrium constant.     

Tyr219 of human matrix metalloproteinase 7 is not critical for catalytic activity, but is involved in the broad pH-dependence of the activity

Human matrix metalloproteinase 7 (MMP-7) exhibits a broad bell-shaped pH-dependence with the acidic and alkaline pK(e) (pK(e1) and pK(e2)) values of about 4 and 10. Its active-site tyrosyl residue, Tyr219, is conserved in all other MMPs, and thus has been thought for the ionizable group responsible for pK(e2). In this study, we examined the mutational effects of Tyr219 on enzyme activity. Five Tyr219 variants, Y219F (Tyr219 is replaced with Phe), Y219D, Y219A, Y219C and Y219S, were constructed by site-directed mutagenesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl-l-Pro-l-Leu-Gly-l-Leu-[N(3)-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl]-l-Ala-l-Arg-NH(2), all five variants retained the activity, indicating that Tyr219 is not the ionizable group responsible for pK(e2). Unexpectedly, all five variants exhibited narrower pH-dependence than the wild-type MMP-7, with the pK(e1) and pK(e2) values in the range of 5.2-5.4 and 8.6-9.4, respectively. Such pH-dependence shifts were not observed in other active-site tyrosyl-residue variants, Y193F and Y216F. These results suggest that Tyr219 is not critical for catalytic activity, but is involved in the broad pH-dependence of the activity.     

Modification of lipoxygenase by hydrogen peroxide and photooxidation.

The kinetic study of fluorescence stopped-flow method suggested that the interaction between lipoxygenase and H2O2 is consistent with a simple irreversible one-step mechanism. The activation energy of the reaction was 7.2 kcal/mol. Participation of an ionizable group with pK about 8.8, possibly a histidine residue, was suggested from the pH-dependence of the rate constant. No further fluorescence quenching of lipoxygenase was observed when the product was added to the lipoxygenase solution before mixing the lipoxygenase and H2O2 solutions. The fluorescence quenching of lipoxygenase by H2O2 was in parallel with the inactivation of the enzyme. Hydroperoxylinoleic acid strongly protects the inactivation of lipoxygenase caused by H2O2. These results are consistent with an interpretation that OH- and/or O- - are produced when the iron of the enzyme is oxidized by H2O2, which in turn will attack some amino acid essential for the enzyme activity. The pH-dependence of the inactivation rate constant of photooxidation of lipoxygenase sensitized by methylene blue indicated that an ionizable group with pK about 8.8 is concerned with the enzymatic activity. In contrast to the inactivation of lipoxygenase by H2O2, the product protected the inactivation of the enzyme by photooxidation only at high concentration.     

Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of the enzymatic hydrolysis of benzylpenicillin]

The equilibrium constant for penicillin amidase-catalyzed hydrolysis of benzylpenicillin(Keg =3.00 +/- 0.24 x 10(-3) M at pH 5.0) and the ionization constants for phenylacetic acid (PAA) and the amino groups of 6-aminopenicillanic acid (6-APA) were determined (4.20 and 4.60 under conditions of the kinetic experiments respectively). The experimental data at pH 6.0 satisfactorily correlated with the theoretical pH-dependence for Keg constructed according to the hypothesis that benzylpenicillin synthesis has a thermodynamic optimum at pH 4.4 equal to a half-sum of the pK values for the carboxylic and amino groups of the PAA and 6-APA respectively.     

Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis

The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.     

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.     

Critical role of asp227 in the photocycle of proteorhodopsin

The photocycle of the proton acceptor complex mutant D227N of the bacterial retinal protein proteorhodopsin is investigated employing steady state pH-titration experiments in the UV-visible range as well as femtosecond-pump-probe spectroscopy and flash photolysis in the visible spectral range. The evaluation of the pH-dependent spectra showed that the neutralization of the charge at position 227 has a remarkable influence on the ground state properties of the protein. Both the pK(a) values of the primary proton acceptor and of the Schiff base are considerably decreased. Femtosecond-time-resolved measurements demonstrate that the general S(1) deactivation pathway; that is, the K-state formation is preserved in the D227N mutant. However, the pH-dependence of the reaction rate is lost by the substitution of Asp227 with an asparagine. Also no significant kinetic differences are observed upon deuteration. This is explained by the lack of a strongly hydrogen-bonded water in the vicinity of Asp97, Asp227, and the Schiff base or a change in the hydrogen bonding of it (Ikeda et al. (2007) Biochemistry46, 5365-5373). The flash photolysis measurements prove a considerably elongated photocycle with pronounced pH-dependence. Interestingly, at pH 9 the M-state is visible until the end of the reaction cycle, leading to the conclusion that the mutation does not only lower the pK(a) of the Schiff base in the unphotolyzed ground state but also prevents an efficient reprotonation reaction.     

The pH-dependence of pepsin-catalysed reactions

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.     

pH-dependence and structure–activity relationships in the papain-catalysed hydrolysis of anilides

The pH-dependence of the Michaelis-Menten parameters for the papain-catalysed hydrolysis of N-acetyl-l-phenylalanylglycine p-nitroanilide was determined. The equilibrium binding constant, K(s), is independent of pH between 3.7 and 9.3, whereas the acylation constant, k(+2), shows bell-shaped pH-dependence with apparent pK(a) values of 4.2 and 8.2. The effect of substituents in the leaving group on the acylation constant of the papain-catalysed hydrolysis of hippuryl anilides and N-acetyl-l-phenylalanylglycine anilides gives rise in both series to a Hammett rho value of -1.04. This indicates that the enzyme provides electrophilic, probably general-acid, catalysis, as well as the nucleophilic or general-base catalysis previously found. A mechanism involving a tetrahedral intermediate whose formation is general-base-catalysed and whose breakdown is general-acid-catalysed seems most likely. The similarity of the Hammett rho values appears to exclude facilitated proton transfer as a means through which the specificity of papain is expressed.     

The reactions of D-glyceraldehyde 3-phosphate with thiols and the holoenzyme of D-glyceraldehyde 3-phosphate dehydrogenase and of inorganic phosphate with the acyl-holoenzyme.

D-Glyceraldehyde 3-phosphate forms adducts with thiols. These adducts, which are presumed to be hemithioacetals, equilibrate rapidly with the unhydrated form of the aldehyde, which is the subtrate for D-glyceraldehyde 3-phosphate dehydrogenase. The adduct provides a substrate buffer system whereby a constant low free aldehyde concentration can be maintained during the oxidation of aldehyde by the enzyme and NAD+. With this system, the kinetics of the association of the aldehyde with the enzyme were examined. The rate profile for this reaction is a single exponential process, showing that all four active sites of the enzyme have equivalent and independent reactivity towards the aldehyde, with an apparent second-order rate constant of 5 X 10(7)M-1-S-1 at pH8.0 and 21 degrees C. The second-order rate constant becomes 8 X 10(7)M-1-S-1 when account is taken of the forward and reverse catalytic rate constants of the dehydrogenase. The pH-dependence of the observed rate constant is consistent with a requirement for the unprotonated form of a group of pK 6.1, which is the pK observed for second ionization of glyceraldehyde 3-phosphate. The rate of phosphorolysis of the acyl-enzyme intermediate during the steady-state oxidative phosphorylation of the aldehyde was studied, and is proportional to the total Pi concentration up to at least 1 mM-Pi at pH 7.5. The pH-dependence of the rate of NADH generation under these conditions can be explained by the rate law d[NADA]/dt = k[acy] holoenzyme][PO4(3-)-A1, where thioester bond, although kinetically indistinguishable rate equations for the reaction are possible. The rates of the phosphorolysis reaction and of the aldehyde-association reaction decrease with increasing ionic strength, suggesting that the active site of the enzyme has cationic groups which are involved in the reaction of the enzyme with anionic substrates.     

The action of trialkyltin compounds on mitochondrial respiration. The effect of pH

1. Inhibition of 2,4-dinitrophenol-stimulated respiration by trialkyltins is dependent on the presence of Cl(-) in the assay medium and is only apparent at acid pH values. It appears to be a result of the Cl(-)-OH(-) exchange mediated by trialkyltins. 2. In a KCl medium at alkaline pH values, the maximum rate of respiration produced by uncouplers is further increased by the presence of trialkyltins. 3. The inhibition of uncoupled succinate oxidation at acid pH values is not reversed by increasing the external substrate concentration, suggesting that depletion of intramitochondrial succinate is not an important factor in the inhibition. 4. It is suggested that the probable explanation for these observations is that in the presence of Cl(-) trialkyltins alter the internal pH to a more acid value and this directly affects the activity of one or more steps in succinate oxidation. 5. The oligomycin-like action of trialkyltins in a Cl(-)-free medium shows considerable pH-dependence over the pH range 6.6-7.6 in the presence of 10mm-phosphate, but very much less pH-dependence in the presence of 1mm-phosphate. 6. The binding of triethyltin to mitochondria shows a pK at pH6.3 and does not change greatly over the pH range 6.6-7.6. 7. It is suggested that the pH-dependence of the oligomycin-like action described by Coleman & Palmer (1971) is the result of the pH-dependence of the formation of a hydrophilic complex between trialkyltins and P(i).     

Interaction of monodispersed and micellar phospholipids with an Agkistrodon halys blomhoffii phospholipase A2, in which the alpha-amino group had been modified to an alpha-keto group

The pH dependence of the binding constant of Ca2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the alpha-amino group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as alpha-amino group. The pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH-dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH-dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799-804), which was assigned as the alpha-amino group. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the intact and the alpha-NH2-modified enzymes was studied by the pH stat method at 25 degrees C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca2+. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C12PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the alpha-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.     

Bindings of Ca2+ and substrate analogs to a cobra venom phospholipase A2 in which the alpha-amino group is modified to an alpha-keto group

The pH dependence of the chemical reaction rate of p-bromophenacyl bromide (BPB) with His 48 of cobra (Naja naja atra) venom phospholipase A2, in which the alpha-NH2 group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 in the absence of Ca2+. The pH-dependence curve was monophasic with a midpoint at pH 7.9, which corresponds to the pK value of His 48 of the alpha-NH2-modified enzyme, whereas the curve for the intact enzyme was biphasic, indicating participation of two ionizable groups with pK values of 7.3 and 8.55 (Teshima et al. (1982) J. Biochem. 91, 1778-1788). These two groups were thus identified as His 48 and the alpha-NH2 group, respectively. The pH dependence of the binding constant of Ca2+ to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by measuring the tryptophyl fluorescence changes. The pH-dependence curve was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 13-20), and it was interpreted in terms of participation of His 48 and Asp 49 (pK 5.4). The absence of participation of the alpha-NH2 group in the Ca2+ binding was thus confirmed. Bindings of monodispersed n-dodecylphosphorylcholine (n-C12PC) and micellar n-hexadecylphosphorylcholine (n-C16PC) to the alpha-NH2-modified enzyme were studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) and tryptophyl fluorescence methods, respectively. The binding constant of the monodispersed substrate was very similar to that for the intact enzyme (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The binding constant of the micellar substrate to the modified enzyme in the presence of Ca2+ was also very similar to that for the intact enzyme-Ca2+ complex (Teshima et al. (1983) J. Biochem. 94, 223-232), and the pH-dependence curve was interpreted in terms of participation of His 48. On the other hand, the binding constant of the micellar substrate to the modified apoenzyme was much smaller than that for the intact apoenzyme. Nevertheless, the pH-dependence curve could be interpreted in terms of participation of His 48 and Asp 49. From these findings, it was concluded that the ionization state of the alpha-NH2 group of cobra venom phospholipase A2 is essentially irrelevant to the bindings of Ca2+ and also of the monodispersed and micellar substrates.     

Chemical mechanism of beta-xylosidase from Trichoderma reesei QM 9414: pH-dependence of kinetic parameters.

The variation of kinetic parameters of beta-xylosidase from Trichoderma reesei QM 9414 with pH was used to elucidate the chemical mechanism of the p-nitrophenyl beta-D-xylopyranoside hydrolysis. The pH-dependence of V and V/K(m) showed that a group on the enzyme with a pK value of 3.20 must be unprotonated and a group with a pK value of 5.20 must be protonated for activity and both are involved in catalysis. Solvent-perturbation studies indicated that these groups are neutral acid type. Temperature dependence of kinetic parameters suggested the stickiness of the substrate at lower temperatures than the optimum and the calculated ionization enthalpies pointed to carboxyl groups as responsible for both pKs. Chemical modification with triethyloxonium tetrafluoroborate and protection with the substrate studies demonstrated essential carboxyl groups on the enzyme. Profiles of pK(i) for D-gluconic acid lactone indicated that a group with a pK value of 3.45 must be protonated for binding and it has been assigned to the carboxyl group of D-gluconic acid formed by lactone ring breakdown in solution.     

Exchange mechanisms for hydrogen bonding protons of cytidylic and guanylic acids.

The pH dependence of buffer catalysis of exchange of the C-4 amino protons of cyclic cytosine 2',3'-monophosphate (cCMP) and the N-1 proton of cyclic guanosine 2',3'-monophosphate (cGMP) conforms to an exchange mechanism, in which protonation of the nucleobases at C(N-3) AND G(N-7) establishes the important intermediates at neutral to acidic pH. Rate constants for transfer of the G(N-1) proton to H2O, OH-, phosphate, acetate, chloracetate, lactate, and cytosine (N-3) were obtained from 1H nuclear magnetic resonance line width measurements at 360 MHz and were used to estimate the pK or acidity of the exchange site in both the protonated and unprotonated nucleobase. These estimates reveal an increase in acidity of the G(N-1) site corresponding to 2 to 3 pK units as the G(N-7) site is protonated: At neutral pH the G(N-1) site of the protonated purine would be ionized (pK = 6.3). Determinations of phosphate, imidazole, and methylimidazole rate constants for transfer of the amino protons of cCMP provide a more approximate estimate of pK = 7 to 9 for the amino of the protonated pyrimidine. A comparison of the intrinsic amino acidity in the neutral and protonated cytosine is vitiated by the observation that OH- catalyzed exchange in the neutral base is not diffusion limited. This leads to the conclusion that protonation of the nucleobase effects a qualitative increase in the ability of the amino protons to form hydrogen bonds: from very poor in the neutral base to "normal" in the conjugate acid.     

pH dependence of progesterone interaction with progesterone-binding globulin. Kinetic and equilibrium studies.

The kinetics of binding and dissociation for the progesterone-binding globulin (PBG)-progesterone complex have been measured as a function of pH. The association rate constant appears to be independent of pH from pH to 10 with an average value of kon = 8.5 X 10(7)M-1 S-1. The dissociation rate constant is strongly pH dependent with the dependency defined by: koff = k0 (1 + [H+]/K1 + K2/[H+])(1 + K3*/[H+])/(1 + K3/[H+]). The best values for the various parameters were k0 = 0.0785 s-1, pK1 = 5.30, pK2 = 10.54, pK3* = 7.41, and pK3 = 7.21. Simpler expressions were inadequate to fit the data, and it was concluded that at least three ionizing residues are responsible for the stability of the PBG-progesterone complex. The affinity constant was determined by equilibrium dialysis over the range of pH 3 to 12. The ratio of the association and dissociation rate constants is in agreement with the affinity constant from pH 6.5 to 10.5. The influence of pH on the conformation and binding activity of PBG was also investigated. Denaturation by acid, base, or guanidine hydrochloride leads to a reversible loss of binding activity. Regain of binding activity in all cases is slow with half-times of 0.5 to 2.7 h, depending on conditions. The rate of acid denaturation was found to be incompletely protonated at pH 1.4, suggesting a buried carboxylic acid residue. The slow renaturation of PBG might be due to the difficulty of burying a charged residue in the protein's interior coupled with steric hindrance by the large carbohydrate moiety of PBG.