Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Catalytic mechanism of the tryptophan synthase alpha(2)beta(2) complex. Effects of pH, isotopic substitution, and allosteric ligands.

The mechanism of the tryptophan synthase alpha(2)beta(2) complex from Salmonella typhimurium is explored by determining the effects of pH, of temperature, and of isotopic substitution on the pyridoxal phosphate-dependent reaction of L-serine with indole to form L-tryptophan. The pH dependence of the kinetic parameters indicates that three ionizing groups are involved in substrate binding and catalysis with pK(a)1 = 6.5, pK(a)2 = 7.3, and pK(a)3 = 8.2-9. A significant primary isotope effect (approximately 3.5) on V and V/K is observed at low pH (pH 7), but not at high pH (pH 9), indicating that the base that accepts the alpha-proton (betaLys-87) is protonated at low pH, slowing the abstraction of the alpha-proton and making this step at least partially rate-limiting. pK(a)2 is assigned to betaLys-87 on the basis of the kinetic isotope effect results and of the observation that the competitive inhibitors glycine and oxindolyl-L-alanine display single pK(i) values of 7.3. The residue with this pK(a) (betaLys-87) must be unprotonated for binding glycine or oxindolyl-L-alanine, and, by inference, L-serine. Investigations of the temperature dependence of the pK(a) values support the assignment of pK(a)2 to betaLys-87 and suggest that the ionizing residue with pK(a)1 could be a carboxylate, possibly betaAsp-305, and that the residue associated with a conformational change at pK(a)3 may be betaLys-167. The occurrence of a closed to open conformational conversion at high pH is supported by investigations of the effects of pH on reaction specificity and on the equilibrium distribution of enzyme-substrate intermediates.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH dependence of penicillin amidase enantioselectivity for charged substrates.

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2). The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis. The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8. The dependence was stronger for the S'(1)- than for the S(1)-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.     

Specific DNA recognition by the type II restriction endonuclease MunI: the effect of pH.

To investigate the effect of pH on sequence-specific binding, a thermodynamic characterization of the interaction of the protein MunI with a specific, and a nonspecific, oligonucleotide was performed. MunI is a type II restriction endonuclease which is able to bind specifically, but loses its enzymatic activity in the absence of magnesium ions. Comparison of the specific and nonspecific interactions at 10 and 25 degrees C shows that the latter is accompanied by a small change in enthalpy, and a negligible change in constant pressure heat capacity. On going through the pH range 5.75-9.0 at 25 degrees C, the affinity of specific complex formation is reduced by 20-fold. The interaction is accompanied by the protonation of groups assumed to be on the protein. Based on the simplest model that will fit the data, two distinct protonation events are observed. At low pH, two groups per protein molecule undergo protonation with a pK(a) of 6.0 and 6.9 in the free and bound forms, respectively. At high pH, a further independent protonation occurs involving two groups with pK(a) values of 8.9 and approximately 10.7 in the free and bound forms, respectively. The change in heat capacity ranges from -2.7 to -1.7 kJ mol(-1) K(-1) in going from pH 6.5 to 8.5. This range of variation of change in heat capacity can be accounted for by the effects of protonation of the interacting molecules. The change in heat capacity, calculated from surface area burial using a previously established relationship (1.15 kJ mol(-1) K(-1)), does not correlate well with the experimentally determined values.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Direct observation of the enthalpy change accompanying the native to molten-globule transition of cytochrome c by using isothermal acid-titration calorimetry

The enthalpy change accompanying the reversible acid-induced transition from the native (N) to the molten-globule (MG) state of bovine cytochrome c was directly evaluated by isothermal acid-titration calorimetry (IATC), a new method for evaluating the pH dependence of protein enthalpy. The enthalpy change was 30 kJ/mol at 30 degrees C, pH 3.54, with 500 mM KCl. The results of the global analysis of the temperature dependence of the excess enthalpy from 20 to 35 degrees C demonstrated that the N to MG transition is a two-state transition with a small heat capacity change of 1.1 kJ K(-1) mol(-1). The present findings were also indicative of the pH dependence of the enthalpy and the heat capacity of the MG state, -13 kJ mol(-1) pH(-1) and -1.0 kJ K(-1) mol(-1) pH(-1), respectively, at 30 degrees C within a pH range from 2 to 3.     

Multiple sugar binding sites in alpha-glucosidase

Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-glucosidase (EC 3.2.1.20). The K(i) values range from 0.38 mM for 6-deoxy-D-glucose (quinovose) to 1.0 M for D-lyxose at pH=6.3 (0.1 M NaCl, 25 degrees ). All the monosaccharides and the three disaccharides (maltose, isomaltose and alpha,alpha-trehalose) were found to be linear competitive inhibitors with respect to alpha-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K(i)=1.8(+/-0.1) mM], one by D-galactose [K(i)=164(+/-11) mM] and one by D-mannose [K(i)=120(+/-9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK(a1)=5.55(+/-0.15), pK(a2)=6.79(+/-0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme-product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.     

Sequence-specific DNA binding by the glucocorticoid receptor DNA-binding domain is linked to a salt-dependent histidine protonation

We used isothermal titration calorimetry in the temperature range 21-25 degrees C to investigate the effect of pH on the calorimetric enthalpy (delta H(cal)) for sequence specific DNA-binding of the glucocorticoid receptor DNA-binding domain (GR DBD). Titrations were carried out in solutions containing 100 mM NaCl, 1 mM dithiothreitol, 5% glycerol by volume, and 20 mM Tris, Hepes, Mops, or sodium phosphate buffers at pH 7.5. A strong dependence of delta H(cal) on the buffer ionization enthalpy is observed, demonstrating that the DNA binding of the GR DBD is linked to proton uptake at these conditions. The apparent increase in the pK(a) for an amino acid side chain upon DNA binding is supported by the results of complementary titrations, where delta H(cal) shows a characteristic dependence on the solution pH. delta H(cal) is also a function of the NaCl concentration, with opposite dependencies in Tris and Hepes buffers, respectively, such that a similar delta H(cal) value is approached at 300 mM NaCl. This behavior shows that the DNA-binding induced protonation is inhibited by increased concentrations of NaCl. A comparison with structural data suggests that the protonation involves a histidine (His451) in the GR DBD, because in the complex this residue is located close to a DNA phosphate at an orientation that is consistent with a charged-charged hydrogen bond in the protonated state. NMR spectra show that His451 is not protonated in the unbound protein at pH 7.5. The pH dependence in delta H(cal) can be quantitatively described by a shift of the pK(a) of His451 from approximately 6 in the unbound state to close to 8 when bound to DNA at low salt concentration conditions. A simple model involving a binding competition between a proton and a Na(+) counterion to the GR DBD-DNA complex reproduces the qualitative features of the salt dependence.     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

Kinetic and chemical mechanism of the dihydrofolate reductase from Mycobacterium tuberculosis

Dihydrofolate reductase from Mycobacterium tuberculosis (MtDHFR) catalyzes the NAD(P)-dependent reduction of dihydrofolate, yielding NAD(P)(+) and tetrahydrofolate, the primary one-carbon unit carrier in biology. Tetrahydrofolate needs to be recycled so that reactions involved in dTMP synthesis and purine metabolism are maintained. In this work, we report the kinetic characterization of the MtDHFR. This enzyme has a sequential steady-state random kinetic mechanism, probably with a preferred pathway with NADPH binding first. A pK(a) value for an enzymic acid of approximately 7.0 was identified from the pH dependence of V, and the analysis of the primary kinetic isotope effects revealed that the hydride transfer step is at least partly rate-limiting throughout the pH range analyzed. Additionally, solvent and multiple kinetic isotope effects were determined and analyzed, and equilibrium isotope effects were measured on the equilibrium constant. (D(2)O)V and (D(2)O)V/K([4R-4-(2)H]NADH) were slightly inverse at pH 6.0, and inverse values for (D(2)O)V([4R-4-(2)H]NADH) and (D(2)O)V/K([4R-4-(2)H]NADH) suggested that a pre-equilibrium protonation is occurring before the hydride transfer step, indicating a stepwise mechanism for proton and hydride transfer. The same value was obtained for (D)k(H) at pH 5.5 and 7.5, reaffirming the rate-limiting nature of the hydride transfer step. A chemical mechanism is proposed on the basis of the results obtained here.     

Acid-base catalysis in the chemical mechanism of inosine monophosphate dehydrogenase

Inosine-5'-monophosphate dehydrogenase (IMPDH) catalyzes the K+-dependent reaction IMP + NAD + H2O --> XMP + NADH + H+ which is the rate-limiting step in guanine nucleotide biosynthesis. The catalytic mechanism of the human type-II IMPDH isozyme has been studied by measurement of the pH dependencies of the normal reaction, of the hydrolysis of 2-chloro-IMP (which yields XMP and Cl- in the absence of NAD), and of inactivation by the affinity label 6-chloro-purine-ribotide (6-Cl-PRT). The pH dependence of the IMPDH reaction shows bell-shaped profiles for kcat and the kcat/Km values for both IMP and NAD, illustrating the involvement of both acidic and basic groups in catalysis. Half-maximal kcat values occur at pH values of 7.2 and 9.8; similar pK values of 6.9 and 9.4 are seen in the kcat/Km profile for NAD. The kcat/Km profile for IMP, which binds first in the predominantly ordered kinetic mechanism, shows pK values of 8.1 and 7.3 for acidic and basic groups, respectively. None of the kinetic pK values correspond to ionizations of the free substrates and thus reflect ionization of the enzyme or enzyme-substrate complexes. The rate of inactivation by 6-Cl-PRT, which modifies the active site sulfhydryl of cysteine-331, increases with pH; the pK of 7.5 reflects the ionization of the sulfhydryl in the E.6-Cl-PRT complex. The pKs of the acids observed in the IMPDH reaction likely also reflect ionization of the cysteine-331 sulfhydryl which adds to C-2 of IMP prior to NAD reduction. The kcat and kcat/Km values for hydrolysis of 2-Cl-IMP show a pK value of 9.9 for a basic group, similar to that seen in the overall reaction, but do not exhibit the ionization of an acidic group. Surprisingly, the rates of 2-Cl-IMP hydrolysis and of inactivation by 6-Cl-PRT are not stimulated by K+, in contrast to the >100-fold K+ activation of the IMPDH reaction. Apparently the enigmatic role of K+ lies in the NAD(H)-dependent segment of the IMPDH reaction. To evaluate the importance of hydrogen bonding in substrate binding, several deamino- and deoxy-analogues of IMP were tested as substrates and inhibitors. Only 2'-deoxy-IMP was a substrate; the other compounds tested were competitive inhibitors with Ki values at most 10-fold greater than the KD for IMP, illustrating the greater importance of hydrogen-bonding interactions in the chemistry of the IMPDH reaction than simply in nucleotide binding.     

Pseudo-enzymatic hydrolysis of 4-nitrophenyl myristate by human serum albumin

Most of the esterase properties of human serum albumin (HSA) are the result of multiple irreversible chemical modifications rather than turnover. The HSA-catalyzed hydrolysis of 4-nitrophenyl myristate (NphOMy) is consistent with the minimum three-step mechanism involving the acyl-enzyme intermediate HSA-OMy: Under all the experimental conditions, values of K(s) (= k(-1)/k(+1)), k(+2), and k(+2)/K(s) determined under conditions where [HSA] ≥ 5 × [NphOMy] and [NphOMy] ≥ 5 × [HSA] match very well each other. The deacylation process is rate limiting in catalysis (i.e., k(+3) < k(+2)) and k(-2)~k(-3)~0 s(-1). The pH dependence of k(+2)/K(s), k(+2), and K(s) reflects the acidic pK(a)-shift of one ionizing group from 8.9 ± 0.2 in NphOMy-free HSA to 6.8 ± 0.3 in the HSA:NphOMy adduct. The HSA-catalyzed hydrolysis of NphOMy is inhibited competitively by diazepam, indicating that Tyr411 is the active-site nucleophile.     

Effects of Ca(2+)-activated calmodulin on neuronal nitric oxide synthase reductase activity and binding of substrates: pH dependence of kinetic parameters.

The pH dependence of basal and calmodulin- (CaM-) stimulated neuronal nitric oxide synthase (nNOS) reduction of 2,6-dichloroindophenol (DCIP) and cytochrome c(3+) was investigated. The wave-shaped log V versus pH profile revealed that optimal DCIP reduction occurred when a group, pK(a) of 7.6-7.8, was ionized. The (V/K)(NADPH) and (V/K)(DCIP) versus pH profiles increased with the protonation of a group with a pK(a) of 6.5 or 5.9 and the ionization of two groups with the same pK(a) of 7.5 or 7.0, respectively. (V/K)(DCIP) decreased with the ionization of a group, pK(a) of 9.0. Similar V, (V/K)(NADPH), and (V/K)(DCIP) versus pH profiles for DCIP reduction were obtained with and without CaM, indicating that CaM does not influence ionizable groups involved in catalysis or substrate binding. In contrast, CaM affected the pH dependence of cytochrome c(3+) reduction. The wave-shaped log V versus pH profile for basal cytochrome c(3+) reduction revealed that ionization of a group, pK(a) of 8.6, increased catalysis. Log V for CaM-stimulated cytochrome c(3+) reduction displayed a bell-shaped pH dependence with the protonation of a group with a pK(a) of 6.4 and the ionization of a group with a pK(a) of 9.3, resulting in a loss of activity. The log(V/K)(cytc) versus pH profiles with and without CaM were bell-shaped with the ionization of a group at pK(a) of 7.1 or 7.6 (CaM) or pK(a) of 9.4 or 9.6 (CaM), increasing and decreasing (V/K)(cytc). These results suggest that CaM may change the nature of the rate-limiting catalytic steps or ionizable groups involved in cytochrome c(3+) reduction.     

Energetics of Ca(2+)-EDTA interactions: calorimetric study

The interaction between Ca(2+) and EDTA has been studied using isothermal titration calorimetry to elucidate the detailed mechanism of complex formation and to relate the apparent thermodynamic parameters of calcium binding to the intrinsic effects of ionization. It has been shown that Ca(2+) binding to EDTA is an exothermic process in the temperature range 5-48 degrees C and is highly dependent on the buffer in which the reaction occurs. Calorimetric measurements along with pH-titration of EDTA under different solvent conditions shows that the apparent enthalpy effect of the binding is predominantly from the protonation of buffer. Subtraction of the ionization effect of buffer from the total enthalpy shows that the enthalpy of binding Ca(2+) to EDTA is -0.56 kcal mol(-1) at pH 7.5. The DeltaH value strongly depends on solvent conditions as a result of the degree of ionization of the two amino groups in the EDTA molecule, but depends little on temperature, indicating that the heat capacity increment for metal binding is close to zero. At physiological pH values where the amino groups of EDTA with pK(a)=6.16 and pK(a)=10.26 are differently ionized, the coordination of the Ca(2+) ion into the complex leads to release of one proton due to deprotonation of the amino group having pK(a)=10.26. Increasing the pH up to 11.2, where little or no ionization occurs, leads to elimination of the enthalpy component due to ionization, while its decrease to pH 2 leads to its increase, due to protonation of the two amino groups. The heat effect of Ca(2+)/EDTA interactions, excluding the deprotonation enthalpy of the amino groups, i.e. that associated with the intrinsic enthalpy of binding, is higher in value (Delta(b)H(o)=-5.4 kcal mol(-1)) than the apparent enthalpy of binding. Thus, the large DeltaG value for Ca(2+) binding to EDTA arises not only from favorable entropic but also enthalpic changes, depending on the ionization state of the amino groups involved in coordination of the calcium. This explains the great variability in DeltaH obtained in previous studies. The ionization enthalpy is always unfavorable, and therefore dramatically decreases Ca(2+) affinity by reduction of the enthalpy term of the stability function. The origin of the enthalpy and entropy terms in the stability of the Ca(2+)-EDTA complex is discussed.     

Stopped-flow kinetic studies of the cellulase-catalyzed conversion of cellulose to glucose

The kinetics of the cellulase-catalyzed conversion of soluble cellulose into glucose have been studied over a range of substrate concentrations and temperatures, and at pH values ranging from 4.75 to 7.0. Lineweaver-Burk plots were linear and led to V = 6.2muM/s and K(m) = 13.1 mM at pH 5.8 and 25.0 degrees C. The pK values corresponding to the free enzyme are 4.8 and 6.8 and are consistent with carboxyl and imidazole groups as the active ionizing species. These pK values were little changed in the enzyme-substrate intermediate that reacts in the ratedetermining step, suggesting that the ionizing groups are still free in this intermediate. The activation energy corresponding to V/K(m) is 80.6 kJ/mol, and that corresponding to V is 38.7 kJ/mol. The corresponding entropies of activation are 21 J K(-1) mol(-1) and -157 J K(-1) mol(-1), respectively.     

Catalytic mechanism of SGAP, a double-zinc aminopeptidase from Streptomyces griseus

The catalytic mechanism underlying the aminopeptidase from Streptomyces griseus (SGAP) was investigated. pH-dependent activity profiles revealed the enthalpy of ionization for the hydrolysis of leucine-para-nitroanilide by SGAP. The value obtained (30 +/- 5 kJ.mol(-1)) is typical of a zinc-bound water molecule, suggesting that the zinc-bound water/hydroxide molecule acts as the reaction nucleophile. Fluoride was found to act as a pure noncompetitive inhibitor of SGAP at pH values of 5.9-8 with a K(i) of 11.4 mM at pH 8.0, indicating that the fluoride ion interacts equally with the free enzyme as with the enzyme-substrate complex. pH-dependent pK(i) experiments resulted in a pK(a) value of 7.0, suggesting a single deprotonation step of the catalytic water molecule to an hydroxide ion. The number of proton transfers during the catalytic pathway was determined by monitoring the solvent isotope effect on SGAP and its general acid-base mutant SGAP(E131D) at different pHs. The results indicate that a single proton transfer is involved in catalysis at pH 8.0, whereas two proton transfers are implicated at pH 6.5. The role of Glu131 in binding and catalysis was assessed by determining the catalytic constants (K(m), k(cat)) over a temperature range of 293-329 degrees K for both SGAP and the E131D mutant. For the binding step, the measured and calculated thermodynamic parameters for the reaction (free energy, enthalpy and entropy) for both SGAP and the E131D mutant were similar. By contrast, the E131D point mutation resulted in a four orders of magnitude decrease in k(cat), corresponding to an increase of 9 kJ.mol(-1) in the activation energy for the E131D mutant, emphasizing the crucial role of Glu131 in catalysis.     

The catalytic role of glutamate 151 in the leucine aminopeptidase from Aeromonas proteolytica

Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K(m)) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k(cat) and K(m) revealed a change in the pK(a) of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK(a) values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.     

The effect of pH on the kinetics of arylsulphatases A and B.

The effect of pH on the kinetics of rat liver arylsulphatases A and B is very similar and shows that two groups with pK values of 4.4-4.5 and 5.7-5.8 are important for enzyme activity. Substrate binding has no effect on the group with a pK of 4.4-4.5; however, the pK of the second group is shifted to 7.1-7.5 in the enzyme-substrate complex. An analysis of the effect of pH on the Ki for sulphate inhibition suggests that HSO4-is the true product. A model is proposed that involves the two ionizing groups identified in the present study in a concerted general acid-base-catalysed mechanism.