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.     

Four states of tyrosine residues in the fibrinogen molecule

The ionization of tyrosine residues in fibrinogen was studied by a spectrophotometric method. The total of 100 tyrosine residues in the fibrinogen molecule was classified into four states: (1) 28 tyrosine residues with pK 10.1 (m = 1.0). (2) tyrosine residues with pK 11.5 (m = 1.0), (3) 20 tyrosine residues with pK 12.2 (m = 3.0) and (4) 10 tyrosine residues non-ionizable. When fibrinogen was treated with 4 M guanidine . HCl, all of the tyrosine residues became ionizable with the ionization characteristics of pK 10.1 (m = 1.0). The ionization characteristics of tyrosine residues in plasmin-digested fibrinogen were similar to those of fibrinogen, while in CNBr-treated fibrinogen they were fairly different. The value, m, stands for the number of hydroxyl ions involved in the ionization of a tyrosine residue.     

Dissecting the electrostatic interactions and pH-dependent activity of a family 11 glycosidase

Previous studies of the low molecular mass family 11 xylanase from Bacillus circulans show that the ionization state of the nucleophile (Glu78, pK(a) 4.6) and the acid/base catalyst (Glu172, pK(a) 6.7) gives rise to its pH-dependent activity profile. Inspection of the crystal structure of BCX reveals that Glu78 and Glu172 are in very similar environments and are surrounded by several chemically equivalent and highly conserved active site residues. Hence, there are no obvious reasons why their apparent pK(a) values are different. To address this question, a mutagenic approach was implemented to determine what features establish the pK(a) values (measured directly by (13)C NMR and indirectly by pH-dependent activity profiles) of these two catalytic carboxylic acids. Analysis of several BCX variants indicates that the ionized form of Glu78 is preferentially stabilized over that of Glu172 in part by stronger hydrogen bonds contributed by two well-ordered residues, namely, Tyr69 and Gln127. In addition, theoretical pK(a) calculations show that Glu78 has a lower pK(a) value than Glu172 due to a smaller desolvation energy and more favorable background interactions with permanent partial charges and ionizable groups within the protein. The pK(a) value of Glu172 is in turn elevated due to electrostatic repulsion from the negatively charged glutamate at position 78. The results also indicate that all of the conserved active site residues act concertedly in establishing the pK(a) values of Glu78 and Glu172, with no particular residue being singly more important than any of the others. In general, residues that contribute positive charges and hydrogen bonds serve to lower the pK(a) values of Glu78 and Glu172. The degree to which a hydrogen bond lowers a pK(a) value is largely dependent on the length of the hydrogen bond (shorter bonds lower pK(a) values more) and the chemical nature of the donor (COOH > OH > CONH(2)). In contrast, neighboring carboxyl groups can either lower or raise the pK(a) values of the catalytic glutamic acids depending upon the electrostatic linkage of the ionization constants of the residues involved in the interaction. While the pH optimum of BCX can be shifted from -1.1 to +0.6 pH units by mutating neighboring residues within the active site, activity is usually compromised due to the loss of important ground and/or transition state interactions. These results suggest that the pH optima of an enzyme might be best engineered by making strategic amino acid substitutions, at positions outside of the "core" active site, that electrostatically influence catalytic residues without perturbing their immediate structural environment.     

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.     

Dependence of the catalytic activity of papain on the ionization of two acidic groups.

The pH dependence of kcat/Km for the papain-catalyzed hydrolysis of ethyl hippurate, N-alpha-benzoyl-L-citrulline methyl ester, and the p-nitroanilide, amide, and ethyl ester derivatives of N-alpha-benzoyl-L-arginine was determined below pH 6.4. The value of kcat/Km was observed to be modulated by two acid ionizations rather than a single ionization as previously believed. For the five substrates studied, the average pK values for the two ionizations are 3.78 +/- 0.2 and 3.95 +/- 0.1 at T/2 0.3, 25 degrees C. The observation that similar pK values were obtained with different substrates was taken as evidence that the kinetically determined pK values are close in value to true macroscopic ionization constants for ionization of groups on the free enzyme.     

Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes

Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.     

pH-dependence of the esterolytic activity of terrylytine proteases

The pH-dependences of the kinetic parameters of hydrolysis of alpha-N-henzoyl-L-arginine and N-benzoyl-L-tyrosine ethyl esters by terrylytine proteases I and II were studied. The experimental results proved evidence for the differences in the activities of these enzymes. The active center of protease I contains two groups with close pK values (6,5-7,0), one of which is presumably the imidazole group of histidine. The catalytic action of protease II is determined by the state of the ionizing groups with the ionization constants of about 6 and 8. The former functions in the deprotonated form, whereas the latter--in the protonated form.     

pK(a) calculations for class C beta-lactamases: the role of Tyr-150

The Poisson-Boltzmann method was used to compute the pK(a) values of titratable residues in a set of class C beta-lactamases. In these calculations, the pK(a) of the phenolic group of residue Tyr150 is the only one to stand out with an abnormally low value of 8.3, more than one pK(a) unit lower than the measured reference value for tyrosine in solution. Other important residues of the catalytic pocket, such as the conserved Lys67, Lys315, His314, and Glu272 (hydrogen-bonded to the ammonium group of Lys315), display normal protonation states at neutral pH. pK(a) values were also computed in catalytically impaired beta-lactamase mutants. Comparisons between the relative k(cat) values and the Tyr150 pK(a) value in these mutants revealed a striking correlation. In active enzymes, this pK(a) value is always lower than the solution reference value while it is close to normal in inactive enzymes. These results thus support the hypothesis that the phenolate form of Tyr150 is responsible for the activation of the nucleophilic serine. The possible roles of Lys67 and Lys315 during catalysis are also discussed.     

Tissue factor alters the pK(a) values of catalytically important factor VIIa residues

Blood coagulation is triggered when the serine protease factor VIIa (fVIIa) binds to cell surface tissue factor (TF) to form the active enzyme-cofactor complex. TF binding to fVIIa allosterically augments the enzymatic activity of fVIIa toward macromolecular substrates and small peptidyl substrates. The mechanism of this enhancement remains unclear. Our previous studies have indicated that soluble TF (sTF; residues 1-219) alters the pH dependence of fVIIa amidolytic activity (Neuenschwander et al. (1993) Thromb. Haemostasis 70, 970), indicating an effect of TF on critical ionizations within the fVIIa active center. The pKa values and identities of these ionizable groups are unknown. To gain additional insight into this effect, we have performed a detailed study of the pH dependence of fVIIa amidolytic activity. Kinetic constants of Chromozym t-PA (MeSO(2)-D-Phe-Gly-Arg-pNA) hydrolysis at various pH values were determined for fVIIa alone and in complex with sTF. The pH dependence of both enzymes was adequately represented using a diprotic model. For fVIIa alone, two ionizations were observed in the free enzyme (pK(E1) = 7.46 and pK(E2) = 8.67), with at least a single ionization apparent in the Michaelis complex (pK(ES1) similar 7.62). For the fVIIa-TF complex, the pK(a) of one of the two important ionizations in the free enzyme was shifted to a more basic value (pK(E1) = 7.57 and pK(E2) = 9.27), and the ionization in the Michaelis complex was possibly shifted to a more acidic pH (pK(ES1) = 6.93). When these results are compared to those obtained for other well-studied serine proteases, K(E1) and K(ES1) are presumed to represent the ionization of the overall catalytic triad in the absence and presence of substrate, respectively, while K(E2) is presumed to represent ionization of the alpha-amino group of Ile(153). Taken together, these results would suggest that sTF binding to fVIIa alters the chemical environment of the fVIIa active site by protecting Ile(153) from deprotonation in the free enzyme while deprotecting the catalytic triad as a whole when in the Michaelis complex.     

Control of coenzyme binding to horse liver alcohol dehydrogenase.

The rate of association of NAD(+) with wild-type horse liver alcohol dehydrogenase (ADH) is maximal at pH values between pK values of about 7 and 9, and the rate of NADH association is maximal at a pH below a pK of 9. The catalytic zinc-bound water, His-51 (which interacts with the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of the coenzyme in the proton relay system), and Lys-228 (which interacts with the adenosine 3'-hydroxyl group and the pyrophosphate of the coenzyme) may be responsible for the observed pK values. In this study, the Lys228Arg, His51Gln, and Lys228Arg/His51Gln (to isolate the effect of the catalytic zinc-bound water) mutations were used to test the roles of the residues in coenzyme binding. The steady state kinetic constants at pH 8 for the His51Gln enzyme are similar to those for wild-type ADH. The Lys228Arg and Lys228Arg/His51Gln substitutions decrease the affinity for the coenzymes up to 16-fold, probably due to altered interactions with the arginine at position 228. As determined by transient kinetics, the rate constant for association of NAD(+) with the mutated enzymes no longer decreases at high pH. The pH profile for the Lys228Arg enzyme retains the pK value near 7. The His51Gln and Lys228Arg/His51Gln substitutions significantly decrease the rate constants for NAD(+) association, and the pH dependencies show that these enzymes bind NAD(+) most rapidly at a pH above pK values of 8. 0 and 9.0, respectively. It appears that the pK of 7 in the wild-type enzyme is shifted up by the H51Q substitutions, and the resulting pH dependence is due to the deprotonation of the catalytic zinc-bound water. Kinetic simulations suggest that isomerization of the enzyme-NAD(+) complex is substantially altered by the mutations. In contrast, the pH dependencies for NADH association with His51Gln, Lys228Arg, and Lys228Arg/His51Gln enzymes were the same as for wild-type ADH, suggesting that the binding of NAD(+) and the binding of NADH are controlled differently.     

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.     

Amino acid residues involved in the catalytic mechanism of NAD-dependent glutamate dehydrogenase from Halobacterium salinarum

The pH dependence of kinetic parameters for a competitive inhibitor (glutarate) was determined in order to obtain information on the chemical mechanism for NAD-dependent glutamate dehydrogenase from Halobacterium salinarum. The maximum velocity is pH dependent, decreasing at low pHs giving a pK value of 7.19+/-0.13, while the V/K for l-glutamate at 30 degrees C decreases at low and high pHs, yielding pK values of 7.9+/-0.2 and 9.8+/-0.2, respectively. The glutarate pKis profile decreases at high pHs, yielding a pK of 9. 59+/-0.09 at 30 degrees C. The values of ionization heat calculated from the change in pK with temperature are: 1.19 x 10(4), 5.7 x 10(3), 7 x 10(3), 6.6 x 10(3) cal mol-1, for the residues involved. All these data suggest that the groups required for catalysis and/or binding are lysine, histidine and tyrosine. The enzyme shows a time-dependent loss in glutamate oxidation activity when incubated with diethyl pyrocarbonate (DEPC). Inactivation follows pseudo-first-order kinetics with a second-order rate constant of 53 M-1min-1. The pKa of the titratable group was pK1=6.6+/-0.6. Inactivation with ethyl acetimidate also shows pseudo-first-order kinetics as well as inactivation with TNM yielding second-order constants of 1.2 M-1min-1 and 2.8 M-1min-1, and pKas of 8.36 and 9.0, respectively. The proposed mechanism involves hydrogen binding of each of the two carboxylic groups to tyrosyl residues; histidine interacts with one of the N-hydrogens of the l-glutamate amino group. We also corroborate the presence of a conservative lysine that has a remarkable ability to coordinate a water molecule that would act as general base.     

Calorimetric and potentiometric characterization of the ionization behavior of ribonuclease A and its complex with 3'-cytosine monophosphate.

The proton association behavior of ribonuclease A and its complex with 3'-cytosine monophosphate has been thermodynamically characterized in the pH range 4--8 at 25 degrees, mu = 0.05. Calorimetric and potentiometric titration data have been used to estimate the apparent pK values and enthalpy values for protonation of the four histidine residues of the protein, deltaHp. In the free enzyme the pK values were deduced to be 5.0, 5.8, 6.6, and 6.7 and deltaHp deduced to be -6.5, -6.5, -6.5, and -24 kcal/mol for residues 119, 12, 105, and 48, respectively. For the nucleotide-enzyme complex it was concluded that the apparent pK values of residues 119, 12, and 48 increased to an average value of about 7.2, the deltaHp values remaining constant for all histidine groups except 48. It was also concluded that only the dianionic phosphate form of the nucleotide inhibitor is bound to the enzyme in this pH range. These results are consistent with a thermodynamic model for the binding reaction in which inhibitor-enzyme association is coupled to the ionization of three imidazole residues (12, 119, and 48) and the interaction between the negative phosphate moiety of the inhibitor and the positively charged residues 12 and 119 is purely electrostatic. However, the "interaction" with residue 48 probably involves a conformational rearrangement of the macromolecule.     

Roles of cysteine 161 and tyrosine 154 in the lecithin-retinol acyltransferase mechanism

Lecithin-retinol acyltransferase (LRAT) catalyzes the transfer of an acyl moiety from the sn-1 position of lecithin to vitamin A, generating all-trans-retinyl esters. LRAT is a unique enzyme and is the founder member of an expanding group of proteins of largely unknown function. In an effort to understand the mechanism of LRAT action, it was of interest to assign the amino acid residues responsible for the two pK(a) values of 8.22 and 9.95 observed in the pH vs rate profile. Titrating C161 of LRAT with a specific affinity labeling agent at varying pH values shows that this residue has a pK(a) = 8.03. Coupled with previous studies, this titration reveals the catalytically essential C161 as the residue responsible for the ascending limb of the pH vs rate profile. Site-specific mutagenic experiments on the lysine and tyrosine residues of LRAT reveal that only the highly conserved tyrosine 154 is essential for catalytic activity. This residue is likely to be responsible for the pK(a) = 9.95 found in the pH vs rate profile. Thus, LRAT has three essential residues (C161, Y154, and H60), all of which are conserved in the LRAT family of enzymes.     

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.     

Structural reorganization triggered by charging of Lys residues in the hydrophobic interior of a protein

Structural consequences of ionization of residues buried in the hydrophobic interior of proteins were examined systematically in 25 proteins with internal Lys residues. Crystal structures showed that the ionizable groups are buried. NMR spectroscopy showed that in 2 of 25 cases studied, the ionization of an internal Lys unfolded the protein globally. In five cases, the internal charge triggered localized changes in structure and dynamics, and in three cases, it promoted partial or local unfolding. Remarkably, in 15 proteins, the ionization of the internal Lys had no detectable structural consequences. Highly stable proteins appear to be inherently capable of withstanding the presence of charge in their hydrophobic interior, without the need for specialized structural adaptations. The extent of structural reorganization paralleled loosely with global thermodynamic stability, suggesting that structure-based pK(a) calculations for buried residues could be improved by calculation of thermodynamic stability and by enhanced conformational sampling.     

Studies on the catalytic residues at the active site of human liver alpha-L-fucosidase

Kinetic studies and chemical modifications were performed on purified human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) in an attempt to identify the catalytic residues at the active site. Plots of log Vmax vs. pH (computer-fitted to a theoretical model) displayed two apparent pK values, of approx. 3.8 and 7.3. The temperature dependence of these pK values yielded heats of ionization of 3 and 0 kcal/mol from Van't Hoff plots for the lower and higher pK values, respectively. Reaction of alpha-L-fucosidase with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and sodium p-(hydroxymercuri)benzoate resulted in complete inactivation of the enzyme. Other nonspecific inactivators had little or no effect on enzyme activity. These results suggest two carboxyl groups whose ionization state is important to activity, a non-active-site cysteine residue important to activity, and at least one active-site carboxyl group.     

Characterization of an essential histidine residue in thermophilic malate dehydrogenase

Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate. The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme. The enzyme was protected from inactivation by NADH. The enzyme was also inactivated by dye-sensitized photooxidation. Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme. Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C. From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation. The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.     

Marcus rate theory applied to enzymatic proton transfer

The hydration of CO(2) and the dehydration of HCO(3)(-) catalyzed by the carbonic anhydrases is accompanied by the transfer of protons between solution and the zinc-bound water molecule in the active site. This transfer is facilitated by amino acid residues of the enzyme which act as intramolecular proton shuttles; variants of carbonic anhydrase lacking such shuttle residues are enhanced or rescued in catalysis by intermolecular proton transfer from donors such as imidazole in solution. The resulting rate constants for proton transfer when compared with the values of the pK(a) of the donor and acceptor give Bronsted plots of high curvature. These data are described by Marcus theory which shows an intrinsic barrier for proton transfer from 1 to 2 kcal/mol and work terms or thermodynamic contributions to the free energy of reaction from 4 to10 kcal/mol. The interpretation of these Marcus parameters is discussed in terms of the well-studied pathway of the catalysis and structure of the enzymes.     

Formation of an RNase A derivative containing an aminosuccinyl residue in place of asparagine 67

At acidic pH, Asp67 and beta-Asp67 (beta-Asp: isoaspartic acid residue) derivatives of RNase A, obtained by selective deamidation of the parent enzyme, spontaneously produces a new derivative containing an aminosuccinyl residue (Asu). The overall secondary structure of the protein chain does not change as a consequence of this substitution, while the catalytic activity on RNA is reduced to about 25%. The pH dependence of the first-order rate constants for the Asu formation has a bell-shaped profile, the maximum being close to the pK(a) of the aspartic acid side chains. Moreover, the values of the rate constants are of the same magnitude of those measured for Asp-containing peptides whose sequence mimics the Asu formation site of the enzyme. This feature indicates that Asp67 and beta-Asp67 residues in the deamidated RNase A derivatives are sited in a region flexible enough to permit the cyclization of the carboxylic side chain to succinimide ring. These results are discussed at the light on to the three-dimensional structure and the thermodynamic stability of the aspartic acid derivatives of RNase A.