The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Arylamidase of Neisseria catarrhalis

Neisseria catarrhalis produces arylamidase intracellularly and is one of the gram-negative bacteria producing exceptionally large amounts of this enzyme.In general, gram-positive bacteria do not produce this enzyme. Arylamidase from N. catarrhalis was purified by salt fractionation, chromatography, and density gradient ultracentrifugation. Its sedimentation coefficient was 6.6; l-alanine-beta-naphthylamide (betaNA) was the most rapidly hydrolyzed amino acid-betaNA. The enzyme had pK(e) values of 6.1 and 8.7 and pK(es) values of 7.1 and 7.9; only those amino acid-betaNA compounds of the l configuration were susceptible to hydrolysis. Arylamidase catalyzed stepwise hydrolysis of dipeptide-betaNA, beginning with the N-terminal residue. Substrates having amino acid residues with larger R groups, such as leucine, interacted much more effectively with enzyme. The significance of the predominate occurrence of arylamidase activity in gram-negative bacteria and the role of this enzyme in the physiology of these organisms remain unclear. It has been established, however, that arylamidase is distinct from leucine aminopeptidase.     

Specificity and catalysis of uracil DNA glycosylase. A molecular dynamics study of reactant and product complexes with DNA.

The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.     

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

The equilibrium parameters of the hydrolysis of ampicillin catalysed by penicillin amidase were determined within the pH range of 4.5 to 5.5. The values of the ionization constants of the carboxy group of D-(-)-ALPHA-AMINOPHENYLACETIC ACID (PK1=1.80) and amino group of 6-aminopenicillanic acid (pK2=4.60) were estimated and pH-dependence of the effective free energy of ampicillin hydrolysis was calculated. It was shown that the thermodynamic optimum of ampicillin synthesis was at 3.20 (the value of the effective free energy under the experimental conditions was 3.27 kcal/mole). The value of the "true", pH-independent free energy of hydrolysis (deltasigma) of the amide bond in the ampicillin molecule was determined to be equal to 9.72 kcal/mole. The thermodynamic parameters of ampicillin and benzylpenicillin hydrolysis were compared. The amino group in the alpha-position of phenylacetic acid was shown to have a significant effect on the values of "true" free energy of hydrolysis of the penicillin amide bond and free ionization energy in the system.     

The role of Tyr248 probed by mutant bovine carboxypeptidase A: insight into the catalytic mechanism of carboxypeptidase A

We have investigated the function of Tyr248 using bovine wild-type CPA and its Y248F and Y248A mutants to find that the K(M) values were increased by 4.5-11-fold and the k(cat) values were reduced by 4.5-10.7-fold by the replacement of Tyr248 with Phe for the hydrolysis of hippuryl-L-Phe (HPA) and N-[3-(2-furyl)acryloyl]-Phe-Phe (FAPP), respectively. In the case of O-(trans-p-chlorocinnamoyl)-L-beta-phenyllactate (ClCPL), an ester substrate, the K(M) value was increased by 2.5-fold, and the k(cat) was reduced by 20-fold. The replacement of Tyr248 with Ala decreased the k(cat) values by about 18- and 237-fold for HPA and ClCPL, respectively, demonstrating that the aromatic ring of Tyr248 plays a critical role in the enzymic reaction. The increases of the K(M) values were only 6- and 5-fold for HPA and ClCPL, respectively. Thus, the present study indicates clearly that Tyr248 plays an important role not only in the binding of substrate but also in the enzymic hydrolysis. The kinetic results may be rationalized by the proposition that the phenolic hydroxyl of Tyr248 forms a hydrogen bond with the zinc-bound water molecule, causing further activation of the water molecule by reducing its pK(a) value. The pH dependency study of k(cat) values and the solvent isotope effects also support the proposition. A unified catalytic mechanism is proposed that can account for the different kinetic behavior observed in the CPA-catalyzed hydrolysis of peptide and ester substrates.     

Determinants of cysteine pKa values in creatine kinase and alpha1-antitrypsin

The structural determinants of the unusually low pK(a) values of Cys282 in human creatine kinase and Cys232 in alpha1-antitrypsin were studied computationally. We have demonstrated that hydrogen bonding to the cysteine residue is the prime determinant for both proteins. In the case of creatine kinase, the hydrogen bond donors are a serine side chain and an amide NH-group, while in alpha1-antitrypsin the donor is an amide NH. Each hydrogen bond lowers the pK(a) by between 0.8 and 1.5 pH units. The 1.1-unit lowering due to the Ser284-Cys282 hydrogen bond is in good agreement with the 1.2-unit difference between the Cys282 pK(a) value of wild-type and the S284A mutant of creatine kinase.     

Adenine protonation in domain B of the hairpin ribozyme

Protein enzymes often use ionizable side chains, such as histidine, for general acid-base catalysis because the imidazole pK(a) is near neutral pH. RNA enzymes, on the other hand, are comprised of nucleotides which do not have apparent pK(a) values near neutral pH. Nevertheless, it has been recently shown that cytidine and adenine protonation can play an important role in both nucleic acid structure and catalysis. We have employed heteronuclear NMR methods to determine the pK(a) values and time scales of chemical exchanges associated with adenine protonation within the catalytically essential B domain of the hairpin ribozyme. The large, adenine-rich internal loop of the B domain allows us to determine adenine pK(a) values for a variety of non-Watson-Crick base pairs. We find that adenines within the internal loop have pK(a) values ranging from 4.8 to 5.8, significantly higher than the free mononucleotide pK(a) of 3. 5. Adenine protonation results in potential charge stabilization, hydrogen bond formation, and stacking interactions that are expected to stabilize the internal loop structure at low pH. Fast proton exchange times of 10-50 micros were determined for the well-resolved adenines. These results suggest that shifted pK(a) values may be a common feature of adenines in non-Watson-Crick base pairs, and identify two adenines which may participate in hairpin ribozyme active site chemistry.     

Detection of large pKa perturbations of an inhibitor and a catalytic group at an enzyme active site, a mechanistic basis for catalytic power of many enzymes

Delta(5)-3-Ketosteroid isomerase catalyzes cleavage and formation of a C-H bond at a diffusion-controlled limit. By determining the crystal structures of the enzyme in complex with each of three different inhibitors and by nuclear magnetic resonance (NMR) spectroscopic investigation, we evidenced the ionization of a hydroxyl group (pK(a) approximately 16.5) of an inhibitor, which forms a low barrier hydrogen bond (LBHB) with a catalytic residue Tyr(14) (pK(a) approximately 11.5), and the protonation of the catalytic residue Asp(38) with pK(a) of approximately 4.5 at pH 6.7 in the interaction with a carboxylate group of an inhibitor. The perturbation of the pK(a) values in both cases arises from the formation of favorable interactions between inhibitors and catalytic residues. The results indicate that the pK(a) difference between catalytic residue and substrate can be significantly reduced in the active site environment as a result of the formation of energetically favorable interactions during the course of enzyme reactions. The reduction in the pK(a) difference should facilitate the abstraction of a proton and thereby eliminate a large fraction of activation energy in general acid/base enzyme reactions. The pK(a) perturbation provides a mechanistic ground for the fast reactivity of many enzymes and for the understanding of how some enzymes are able to extract a proton from a C-H group with a pK(a) value as high as approximately 30.     

Roles of the disulfide bond and adjacent residues in determining the reduction potentials and stabilities of respiratory-type Rieske clusters

Rieske [2Fe-2S] clusters have reduction potentials which vary by over 500 mV, and which are pH dependent. In the cytochrome bc(1) complex, the high-potential and low-pK values of the cluster may be important in the mechanism of quinol oxidation. Hydrogen bonds, from both side-chain and mainchain groups, are crucial for these properties, but solvent accessibility and a disulfide bond (present in only high-potential Rieske proteins) have been suggested to be important determinants also. Previous studies have addressed the hydrogen bonds, disulfide bond, and a leucine residue which may restrict solvent access, by mutations in the cytochrome bc(1) complex. However, influences on the complex (disruption of quinol binding and displacement of the Rieske domain) are difficult to deconvolute from intrinsic effects on the Rieske cluster. Here, the effects of similar mutations on cluster potential, pK values, and stability are characterized comprehensively in the isolated Rieske domain of the bovine protein. Hydrogen bonds from Ser163 and Tyr165 are important in increasing the reduction potential and decreasing the pK values. The disulfide has a limited effect on the redox properties, but is crucial for cluster stability, particularly in the oxidized state. Mutations of Leu142 had little effect on cluster potential, pK values, or stability, in contrast to the significant effects which were observed in the complex. The sum of the effects of all the mutated residues accounts for most of the differences between high- and low-potential Rieske proteins.     

Substrate- and alkali-metal-ion-induced pK shifts in intestinal brush-border sucrase, according to the three-protons model.

To define adequately enzyme activation/inhibition mechanisms as a function of pH, it is necessary to characterize the effector-induced pK shifts on both the free enzyme and on the enzyme-substrate complex. On the basis of our recent three-protons model for sucrase [Vasseur, van Melle, Frangne & Alvarado (1988) Biochem. J. 251, 667-675], we show how the 'fundamental' pK values, deduced from the classical double-logarithmic transformations, are insufficient to generate the required information. This insufficiency derives from the fact that, for sucrase, the acid ionization constant, K1, is a molecular constant that involves complex, V-type plus K-type, activatory and inhibitory kinetic effects. As a consequence, substrate-induced pK shifts cannot be interpreted correctly only by using the fundamental pK approach, because an unequal number of key protons is involved, depending on whether the free enzyme or the enzyme-substrate complex is considered. We demonstrate how this problem can be solved by using the 'theoretical' pK values, derived from the reciprocals of the Michaelis pH functions, i.e. Cha's fractional concentration factors. The procedure we propose, which is general, has the advantage of yielding all the macroscopic pK values for any given model, as calculated from the microscopic pK values. Furthermore, it permits predicting pK shifts as a function of [S] and/or [A] (where S is the substrate and A is the allosteric modifier), an objective that cannot be attained by using the double-logarithmic plot approach. Finally, we describe the relation existing between the fundamental and the theoretical pK values.     

Kinetics of beta-casein hydrolysis by wild-type and engineered trypsin

Apparent rate constants of tryptic hydrolysis of amide bonds containing Arg and Lys residues in beta-casein were determined by the analysis of kinetics of accumulation of 17 major peptide components revealed by high performance liquid chromatography. When studying pH influence on Arg/Lys bond cleavage preference, averaged rate constants over several Arg&bond;X and Lys&bond;X bonds were used for analysis of kinetics of wild-type trypsin, K188H, K188F, K188Y, K188W, and of K188D/D189K mutants. The pK(a1) value of 6.5 was found for all studied trypsins. For wild-type trypsin and its K188D/D189K mutant, pK(a2) was found to be 10. The lowest among studied engineered trypsins pK(a2) = 9.3 was determined for K188Y mutant. Considerable preference for the cleavage of Arg over Lys containing peptide bonds was demonstrated for all trypsins with engineered S2 site except for K188H and K188F. The comparison of individual rate constants for various bonds showed that during the hydrolysis by wild-type trypsin, the probabilities of splitting depend on secondary specificity and local hydrophobicity of amino acid residues, which are nearest to the hydrolyzed peptide bond (P2 site). The improvement of prediction of hydrolysis rates performed by the used program was achieved after considering the presence of hydrophobic neighborhood of Lys48--Ile49 and Arg202--Gly203 bonds.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

Proton-linked subunit heterogeneity in ferrous nitrosylated human adult hemoglobin: an EPR study

The effect of pH on the X-band electron paramagnetic resonance (EPR) spectrum of ferrous nitrosylated human adult tetrameric hemoglobin (HbNO) as well as of ferrous nitrosylated monomeric alpha- and beta-chains has been investigated, at -163 degrees C. At pH 7.3, the X-band EPR spectrum of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains displays a rhombic shape. Lowering the pH from 7.3 to 3.0, tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains undergo a transition towards a species characterized by a X-band EPR spectrum with a three-line splitting centered at 334mT. These pH-dependent spectroscopic changes may be taken as indicative of the cleavage, or the severe weakening, of the proximal HisF8-Fe bond. In tetrameric HbNO, the pH-dependent spectroscopic changes depend on the acid-base equilibrium of two apparent ionizing groups with pK(a) values of 5.8 and 3.8. By contrast, the pH-dependent spectroscopic changes occurring in ferrous nitrosylated monomeric alpha- and beta-chains depend on the acid-base equilibrium of one apparent ionizing group with pK(a) values of 4.8 and 4.7, respectively. The different pK(a) values for the proton-linked spectroscopic transition(s) of tetrameric HbNO and ferrous nitrosylated monomeric alpha- and beta-chains suggest that the quaternary assembly drastically affects the strength of the proximal HisF8-Fe bond in both subunits. This probably reflects a 'quaternary effect', i.e., structural changes in both subunits upon tetrameric assembly, which is associated to a relevant variation of functional properties (i.e., proton affinity).     

Biochemical basis for hydrolysis of organophosphorus by a marine bacterial prolidase

Extensive application of synthesized organophosphorus compounds (OPs) leads to pollutant accumulation and enhanced eco-toxicity. Hydrolysis of phosphotriester bonds catalyzed by evolved microbial enzymes is a key step for detoxification of OPs. Here, a new marine bacterial prolidase OPAA4301 exhibiting promiscuous phosphotriesterase activity was isolated and systematically characterized. The homo-tetrameric enzyme OPAA4301 can catalyze the hydrolysis of both amido bond and phosphotriester bond. Manganese ions were observed to be essential for its catalytic integrity, and in vitro substitution of manganese ions by different metal cofactors led to decreased activity. We also revealed cooperation pattern of metal ligands and substrate-binding residues on OP hydrolysis by mutational analysis. Metal-binding sites together with Arg418 in the large-binding pocket of the enzyme were found to be indispensable for catalytic ability. Substitution mutation of small- and large-binding pocket residues caused significant variation in phosphotriesterase activity, and leaving group sites appeared to be involved in the catalytic process as substrate affinity regulators. Our study gave an overall biochemical understanding on the organophosphorus hydrolysis pattern of the newly identified marine bacterial prolidase and provided ideas for protein engineering to expand its application in the bioremediation field.     

Empirical relationships between protein structure and carboxyl pKa values in proteins

Relationships between protein structure and ionization of carboxyl groups were investigated in 24 proteins of known structure and for which 115 aspartate and 97 glutamate pK(a) values are known. Mean pK(a) values for aspartates and glutamates are < or = 3.4 (+/-1.0) and 4.1 (+/-0.8), respectively. For aspartates, mean pK(a) values are 3.9 (+/-1.0) and 3.1 (+/-0.9) in acidic (pI < 5) and basic (pI > 8) proteins, respectively, while mean pK(a) values for glutamates are approximately 4.2 for acidic and basic proteins. Burial of carboxyl groups leads to dispersion in pK(a) values: pK(a) values for solvent-exposed groups show narrow distributions while values for buried groups range from < 2 to 6.7. Calculated electrostatic potentials at the carboxyl groups show modest correlations with experimental pK(a) values and these correlations are not improved by including simple surface-area-based terms to account for the effects of desolvation. Mean aspartate pK(a) values decrease with increasing numbers of hydrogen bonds but this is not observed at glutamates. Only 10 pK(a) values are > 5.5 and most are found in active sites or ligand-binding sites. These carboxyl groups are buried and usually accept no more than one hydrogen bond. Aspartates and glutamates at the N-termini of helices have mean pK(a) values of 2.8 (+/-0.5) and 3.4 (+/-0.6), respectively, about 0.6 units less than the overall mean values.     

Kinetic studies of urokinase-catalysed hydrolysis of 5-oxo-L-prolylglycyl-L-arginine 4-nitroanilide

The enzymic properties of urokinase (EC 3.4.21.31) were studied. The kinetic parameters of hydrolysis of 5-oxo-Pro-Gly-Arg-NA were determined in the pH range 5-9, at 25 degrees C and 37 degrees C. The reaction is affected by only one ionizing group of urokinase with pK 7.15 (25 degrees C) and pK 6.82 (37 degrees C). The results indicate that 5-oxo-Pro-Gly-Arg-NA is a good model substrate for studies of the conversion of plasminogen to plasmin. The Km values of the urokinase-catalysed hydrolysis of plasminogen and 5-oxo-Pro-Gly-Arg-NA are of the same order of magnitude. Plasmin catalyses the hydrolysis of 5-oxo-Pro-Gly-Arg-NA, but the Km value is several hundred times that of urokinase. Urokinase is shown not to react with good plasmin substrates, such as Bz-Arg-OEt and D-Val-Leu-Lys-NA, but is linearly competitively inhibited by 6-amino-hexanoic acid and trans-4-aminomethylcyclohexane-1-carboxylic acid.     

Determination of dissociation constants of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] in water and biological fluids by means of nuclear magnetic resonance spectroscopy and the DISCO software package

The pK(A) values of (4RS)-[4-carboxy-5,8,11-tris(carboxymethyl)-1-phenyl-2-oxa-5,8,11-triazatridecan-13-oic acid] (BOPTA), a polyprotic molecule whose gadolinium complex is an important magnetic resonance imaging contrast agent for clinical use, have been determined in water, in physiologic solution (PS), in serum (S), and in cerebrospinal fluid (CSF), by means of 13C nuclear magnetic resonance spectroscopy data processed by a dedicated software package called DISCO. The aim of this study was to supply the BOPTA pK(A) values in media very similar to the in vivo environment and, consequently, to get a picture of the in vivo behavior of its Gd complex, whose thermodynamic stability is directly linked to the pK(A) values. The pK(A) values appeared to be almost equal both in D(2)O and in PS, while pK(1) and pK(5) values in CSF differ a little. In S, only pK(2) and pK(3) were calculated due to the narrow pH range used for data collection. However, these pK(A) values were found equal to those in the other media. These results represent the first direct spectroscopic evidence of a substantial invariability of BOPTA behavior in different media and they justify the extrapolation to biological fluids of the data obtained in water. The values also confirmed the high-quality performance of DISCO in calculating pK(A) values of polyprotic molecules in complex media.     

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.     

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.     

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.