Effect of pH on coenzyme binding to liver alcohol dehydrogenase.

1. The transient-state kinetics of ligand-displacement reactions have been analyzed. Methods based on this analysis have been used to obtain reliable estimates of on-velocity and off-velocity constants for coenzyme binding to liver alcohol dehydrogenase at different pH values between 6 and 10. 2. The rate of NADH dissociation from the enzyme shows no pronounced dependence on pH. The rate of NAD+ dissociation is controlled by a group with a pKa of 7.6, agreeing with the pKa reported to regulate the binding of certain inhibitory substrate analogues to the enzyme . NAD+ complex. 3. Critical experiments have been performed to test a recent proposal that on-velocity constants for the binding of NADH and NAD+ are controlled by proton equilibria exhibiting different pKa values. The results show that association rates for NADH and NAD+ exhibit the same pH dependence corresponding to a pKa of 9.2. Titrimetric evidence is presented indicating that the latter effect of pH derives from ionization of a group which affects the anion-binding capacity of the coenzyme-binding site.     

Synergism between coenzyme and alcohol binding to liver alcohol dehydrogenase

Heterotropic cooperativity effects in the binding of alcohols and NAD+ or NADH to liver alcohol dehydrogenase have been examined by equilibrium measurements and stopped-flow kinetic studies. Equilibrium data are reported for benzyl alcohol, 2-chloroethanol, 2,2-dichloroethanol, and trifluoroethanol binding to free enzyme over the pH range 6-10. Binary-complex formation between enzyme and alcohols leads to inner-sphere coordination of the alcohol to catalytic zinc and shows a pH dependence reflecting the ionization states of zinc-bound water and the zinc-bound alcohol. The affinity of the binding protonation state of the enzyme for unionized alcohols increases approximately by a factor of 10 on complex formation between enzyme and NAD+ or NADH. The rate and kinetic cooperativity with coenzyme binding of the alcohol association step indicates that enzyme-bound alcohols participate in hydrogen bonding interactions which affect the rates of alcohol and coenzyme equilibration with the enzyme without providing any pronounced contribution to the net energetics of alcohol binding. The pKa values determined for alcohol deprotonation at the binary-complex level are linearly dependent on those of the free alcohols, and can be readily reconciled with the pKa values attributed to ionization of zinc-bound water. Alcohol coordination to catalytic zinc provides a major contribution to the pKa shift which ensures that the substrate is bound predominantly as an alcoholate ion in the catalytically productive ternary complex at physiological pH. The additional pKa shift contributed by NAD+ binding is less pronounced, but may be of particular mechanistic interest since it increases the acidity of zinc-bound alcohols relatively to that of zinc-bound water.     

Substrate specificity and protonation state of Escherichia coli ornithine transcarbamoylase as determined by pH studies. Binding of carbamoyl phosphate

Binding of carbamoyl phosphate to Escherichia coli ornithine transcarbamoylase and its relation to turnover have been examined as a function of pH under steady-state conditions. The pH profile of the dissociation constant of carbamoyl phosphate (Kiacp) shows that the affinity of the substrate increases as pH decreases. Two ionizing groups are involved in carbamoyl phosphate binding. Protonation of an enzymic group with pKa 9.6 results in productive binding of the substrate with a moderate affinity of Kiacp approximately 30 microM. Protonation of a second group further enhances binding by roughly another order of magnitude. This ionization occurs with a pKa that shifts from less than 6 in the free enzyme to 7.3 in the binary complex. However, tighter binding of carbamoyl phosphate due to this ionization does not contribute to catalysis. The turnover rate (kcat) of the enzyme diminishes in the acidic pH range and is governed by an ionization with a pKa of 7.2. Both the catalytic pKa of 7.2 and the productive binding pKa of 9.6 appear in the pH profile of kcat/KMcp. Together with earlier kinetic results (Kuo, L. C., Herzberg, W., and Lipscomb, W. N. (1985) Biochemistry 24, 4754-4761), these data suggest that the step which modulates kcat may occur prior to the binding of the second substrate L-ornithine.     

4-nitroimidazole binding to horse metmyoglobin: evidence for preferential anion binding

The ionization of 4-nitroimidazole to 4-nitroimidazolate was investigated as a function of ionic strength. The apparent pKa varies from 8.99 to 9.50 between 0.001 and 1.0 M ionic strength, respectively, at 25 degrees C. The ionic strength dependence of this ionization is anomalous. The binding of 4-nitroimidazole by horse metmyoglobin was studied between pH 5.0 and 11.5 and as a function of ionic strength between 0.01 and 1.0 M. The association rate constant is pH-dependent, varying from 24 M(-1)s(-1) at pH 5 to a maximum value of 280 M(-1)s(-1) at pH 9.5 and then decreasing to 10 M(-1)s(-1) at pH 11.5 in 0.1 M ionic strength buffers. The dissociation rate constant has a much smaller pH dependence, varying from 0.082 s(-1) at low pH to 0.035 s(-1) at high pH, with an apparent pKa of 6.5. The binding affinity of 4-nitroimidazole to horse metmyoglobin is about 2.5 orders of magnitude stronger than that for imidazole and this increased affinity is attributed to the much slower dissociation rate for 4-nitroimidazole compared to that of imidazole. Although the ionic strength dependence of the binding rate is small and secondary kinetic salt effects can account for the ionic strength dependence of the association rate constant, the pH dependence of the rate constants and microscopic reversibility arguments indicate that the anionic form of the ligand binds more rapidly to all forms of metmyoglobin than does the neutral form of the ligand. However, the spectrum of the complex is similar to model complexes involving neutral imidazole and not imidazolate. The latter observation suggests that the initial metmyoglobin/4-nitroimidazolate complex rapidly binds a proton and the neutral form of the bound ligand is stabilized, probably through hydrogen binding with the distal histidine.     

Mechanism of binding of horse liver alcohol dehydrogenase and nicotinamide adenine dinucleotide

The binding of NAD+ to liver alcohol dehydrogenase was studied by stopped-flow techniques in the pH range from 6.1 to 10.9 at 25 degrees C. Varying the concentrations of NAD+ and a substrate analogue used to trap the enzyme-NAD+ complex gave saturation kinetics. The same maximum rate constants were obtained with or without the trapping agent and by following the reaction with protein fluorescence or absorbance of a ternary complex. The data fit a mechanism with diffusion-controlled association of enzyme and NAD+, followed by an isomerization with a forward rate constant of 500 s-1 at pH 8: E E-NAD+ *E-NAD+. The isomerization may be related to the conformational change determined by X-ray crystallography of free enzyme and enzyme-coenzyme complexes. Overall bimolecular rate constants for NAD+ binding show a bell-shaped pH dependence with apparent pK values at 6.9 and 9.0. Acetimidylation of epsilon-amino groups shifts the upper pK to a value of 11 or higher, suggesting that Lys-228 is responsible for the pK of 9.0. Formation of the enzyme-imidazole complex abolishes the pK value of 6.9, suggesting that a hydrogen-bonded system extending from the zinc-bound water to His-51 is responsible for this pK value. The rates of isomerization of E-NAD+ and of pyrazole binding were maximal at pH below a pK of about 8, which is attributable to the hydrogen-bonded system. Acetimidylation of lysines or displacement of zinc-water with imidazole had little effect on the rate of isomerization of the E-NAD+ complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

A simple electrostatic model can explain the effect of pH upon the force-pCa relation of skinned frog skeletal muscle fibers.

The relative force-pCa relation of skinned frog skeletal muscle fibers is shifted along the pCa axis by changes in pH. This shift has been interpreted as arising from competition between H+ and Ca2+ for a binding site on troponin. Unfortunately, binding studies have been unable to confirm such competition. Alternatively, however, the data fit a model where H+ influences the degree of dissociation of ionizable groups on the surface of the thin filaments, thus altering the electrostatic potential surrounding the filaments. Alterations in the potential will, in turn, change the concentration of Ca2+ near the troponin binding sites in accordance with the Boltzmann relation. A simple model, based upon the Gouy-Chapman relation between surface potential and charge density, provides a quantitative explanation for the shift of the relative force-pCa curve with pH, given a reasonable estimate of the surface charge density on the thin filament. A best fit is obtained when the ionizable groups giving rise to the potential have a log proton ionization constant (pKa) of 6.1, similar to that for the imidazole group on histidine, and when the density of these groups is near that estimated from amino acid analysis of thin filament proteins and from filament geometry. In preliminary experiments, reaction of skinned frog fibers with diethylpyrocarbonate (DEP) at pH 6 shifted the force-pCa curve toward lower Ca2+. This would be expected in the model since DEP at pH 6 is reported to specifically react with histidine imidazole groups and to irreversibly decrease their pKa, which would increase the net negative charge of the filaments.     

The pH-dependence of the binding of dihydrofolate and substrate analogues to dihydrofolate reductase from Escherichia coli

The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.     

Construction and evaluation of the kinetic scheme associated with dihydrofolate reductase from Escherichia coli

A kinetic scheme is presented for Escherichia coli dihydrofolate reductase that predicts steady-state kinetic parameters and full time course kinetics under a variety of substrate concentrations and pHs. This scheme was derived from measuring association and dissociation rate constants and pre-steady-state transients by using stopped-flow fluorescence and absorbance spectroscopy. The binding kinetics suggest that during steady-state turnover product dissociation follows a specific, preferred pathway in which tetrahydrofolate (H4F) dissociation occurs after NADPH replaces NADP+ in the ternary complex. This step, H4F dissociation from the E X NADPH X H4F ternary complex, is proposed to be the rate-limiting step for steady-state turnover at low pH because koff = VM. The rate constant for hydride transfer from NADPH to dihydrofolate (H2F), measured by pre-steady-state transients, has a deuterium isotope effect of 3 and is rapid, khyd = 950 s-1, essentially irreversible, Keq = 1700, and pH dependent, pKa = 6.5, reflecting ionization of a single group in the active site. This scheme accounts for the apparent pKa = 8.4 observed in the steady state as due to a change in the rate-determining step from product release at low pH to hydride transfer above pH 8.4. This kinetic scheme is a necessary background to analyze the effects of single amino acid substitutions on individual rate constants.     

Proton magnetic relaxation of aspartate transcarbamylase - succinate complexes.

Nuclear magnetic relaxation methods were used to investigate the interaction of the inhibitor succinate with aspartate transcarbamylase from Escherichia coli. Over the pH range 7 to 9, the dissociation constant for succinate remains less than the inhibitor concentration used for most of this work (0.05 M). As a result, the enzyme predominantly exists in a single "gross" conformational state. Succinate binding to this enzyme state (generally known as the R form) parallels the behavior seen previously with the isolated catalytic subunit (Beard, C. B., and Schmidt, P.G. (1973) Biochemistry 12, 2255-2264). The pH and temperature dependence of succinate proton relaxation rates, 1/T2 - 1/T1, in the presence of carbamyl phosphate, is interpreted in terms of a binding mechanism involving three forms of the enzyme, differing by their states of protonation. The least protonated form of the enzyme does not interact with succinate, the singly protonated species binds succinate to form a rapidly dissociating complex, and the doubly protonated species undergoes a conformational isomerization upon succinate binding, yielding a slow exchange complex. Relaxation data provide sufficient information to determine pKa values of 7.2 and 8.9 for two ionizing groups, as well as the dissociation constant for succinate in the fast exchange complex, Kd =1.6 X 10(-2) M. Rate constants for the forward and reverse steps of the isomerization, 1.3 X 10(3) s-1 and 33 s-1, respectively, indicate a significantly slower reverse rate from that obtained in the earlier NMR study of the isolated catalytic subunit. In experiments where the succinate concentration was varied, the relaxation rates showed sigmoidal binding of that ligand to the fast exchange complex above pH 9.1, (a) indicating cooperative binding of succinate, and (b) suggesting that above pH 9.1, the system cannot be characterized by a single dissociation constant, ionization constant, or relaxation effect. CTP and ATP were tested for their ability to affect succinate binding to the fast exchange complex. Heterotropic interactions were observed for CTP but not for ATP. Addition of low concentrations of the transition state analog N-(phosphonacetyl)-L-aspartate to the enzyme-carbamyl phosphate-succinate complex sharply decreased the relaxation rate, indicating that the measurements are sensitive only to succinate bound specifically to the active site.     

Reaction of bovine-liver copper-zinc superoxide dismutase with hydrogen peroxide. Evidence for reaction with H2O2 and HO2- leading to loss of copper

The reaction of hydrogen peroxide with the copper-zinc bovine-liver superoxide dismutase at low molar ratios (0.2-20.0) of H2O2/active site between pH 7.3-10.0 leads to the loss of native enzyme as a distinct form monitored by electrophoresis. The pH dependence of the loss of native enzyme between 7.3 and 9.0 indicates the involvement of a conjugate base on the enzyme of pKa of 8.7 +/- 0.1. The rate of loss of the native enzyme is first order with respect to the concentration of both enzyme and hydrogen peroxide between pH 7.3 and 9.0 with no evidence for binding of peroxide. A second-order rate constant of 3.0 +/- 1.0 M-1 s-1 is obtained from these data. At pH 10.0 the reaction is first order with respect to enzyme concentration but saturable in H2O2. All data are consistent with the interpretation that H2O2 reacts with the enzyme at the lower pH where the reaction is dependent upon the conjugate base of a functional group on the enzyme. At the higher pH, the data are consistent with the reaction of HO2- and H2O2 with the dismutase. The dissociation constant for HO2- calculated from the kinetic data at pH 10.0 is between 25-50 microM and the rate constant for the breakdown of the HO2- dismutase complex is 1.10 + 0.05 x 10(-2) s-1. The change in the electrophoretic pattern at all pH values is accompanied by the loss of the ability of the enzyme to bind copper. Weakly bound or free copper can be detected using bathocuproine disulfonate. Furthermore copper-defficient forms of the enzyme can be detected by staining gels of the peroxide-treated dismutase with diethyldithiocarbamate.     

Characterization of the active site of homogeneous thyroid purine nucleoside phosphorylase.

Purine nucleoside phosphorylase (purine-nucleoside : orthophosphate ribosyltransferase, EC 2.4.2.1) has been purified approx. 4000-fold and to electrophoretic homogeneity from bovine thyroid glands. The isolated enzyme has a specific activity of 17 mumol . min-1 . mg-1. The native enzyme appears to have a molecular weight of 92 000 as determined by sedimentation equilibrum ultracentrifugation and is comprised of three subunits having a molecular weight of 31 000 each as shown by sodium dodecyl sulfate gel electrophoresis. The enzyme is irreversibly denatured below pH 5 and the enzyme-substrate complex is shown to have an ionization constant (pKa) of 9.2 which influences catalytic activity. The pH dependence of the kinetic constants identifies three amino acid ionizable protons. The binding of inosine is effected by an imidazole ring of histidine (pKa 5.65) and a sulfhydryl group of cysteine (pKa 8.5) and the maximal velocity is restricted by an epsilon-amino group which is essential for phosphate binding. The requirement of these residues for activity was confirmed by group-specific chemical modification. The presence of phosphate protected only the lysyl residue while inosine protected all three residues from chemical titration. A model is proposed for the catalytic mechanism of purine nucleoside phosphorylase.     

Interaction of the unique competitive inhibitor imidazole and related compounds with the active site metal of carbonic anhydrase: linkage between pH effects on the inhibitor binding affinity and pH effects on the visible spectra of inhibitor complexes with the cobalt-substituted enzyme

Previous studies on the interaction of carbonic anhydrase (CA) with the unique CO2 competitive inhibitor imidazole and related compounds were all interpreted as showing that an ionizable water ligand on the metal of this zinc metalloenzyme is not displaced by inhibitor binding. Internal inconsistencies in the pH dependence of binding and the pH dependence of the visible spectra of complexes with cobalt-substituted enzyme prompted us to reinvestigate this binding. Visible spectroscopy was used to measure the binding of imidazole and 1,2,4-triazole to Co(II)-substituted human CA I and active site carboxymethylated human CA I (CmCA I) and the binding of 1,2,4-triazole to bovine CoIICA II. The limiting visible spectra for these enzyme-inhibitor adducts were also computed and examined for pH dependence. It was shown that the pKa of visible spectral changes can be independently predicted from studies on the pH dependence of binding. After consideration of possible contributions from effects of His-200 ionization in CA I and CmCA I, and His-64 in CA II, the pH effects on binding affinity and spectra were found to be of the correct magnitude to establish linkage between binding and an ionization. It was also shown, however, that pH effects on binding and spectra cannot distinguish whether neutral imidazole binds to both ionization forms of the enzyme (Zn-OH2 and Zn-OH) or whether neutral imidazole and its anion both bind to only the acid form of the enzyme, presumably after displacing the water. These findings have implications to the crystallographic interpretations on the imidazole-enzyme complex and to the catalytic mechanism of CO2 hydration.     

Quenching of the intrinsic fluorescence of liver alcohol dehydrogenase by the alkaline transition and by coenzyme binding

The fluorescence of alcohol dehydrogenase is quenched by the acid dissociation of some group on the protein having an apparent pKa of 9.6 at 25 degrees C. The pKa of this alkaline quenching transition is unchanged by the binding of trifluoroethanol or pyrazole to the enzyme or by the selective removal of the active site of Zn2+ ion. This indicates that the ionization of a zinc-bound water molecule is not responsible for the quenching. The binding of NAD+ to the enzyme causes a drop in protein fluorescence and an apparent shift in the alkaline quenching transition to lower pH. In the ternary complex formed with NAD+ and trifluoroethanol the alkaline transition is difficult to discern between pH 6 and pH 11. In the NAD+-pyrazole ternary complex, however, a small but noticeable fluorescence transition is observed with a pKa(app) approximately 9.5. We propose that the alkaline transition centered at pH 9.6 is not shifted to lower pH upon binding NAD+. Instead, the amplitude of the alkaline quenching effect is decreased to the point that it is difficult to detect when NAD+ is bound. We present a model that describes the dependence of the fluorescence of the protein on pH and NAD+ concentration in terms of two independently operating, dynamic quenching mechanisms. Our data and model cast serious doubt on the identification, made previously in the literature, between the alkaline quenching pKa and the pKa of the group whose ionization is coupled to NAD+ binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reversible titration of tyrosyl residues in human deoxyhemoglobin

The reaction of hemoglobin with N-acetyl imidazole at neutral pH indicated that in carboxyhemoglobin 1.80 residues per heme were acetylated while in deoxyhemoglobin only 1.15 residues were available to the reagent. The reversible titration of these residues in alkali was followed by difference spectrophotometry at 245 nm. Hill plots of the titration data, assuming 2 residues titrable per heme an3 Δε = 10500 per tyrosyi residue upon ionization, showed a slope of 1.5 and a pH near 11. The average pK of these groups in carboxyhemoglobin was previously found to be near 10.5. Also. by difference spectrophotometry it was shown that exposure of deoxyhemoglobin to alkaline pH was accompanied by a modification of the Soret region of the absorption spectrum, which might indicate the appearance of liganded conformation in the deoxyhemoglobin system. The sedimentation velocity of deoxyhemoglobin demonstrated that at alkaline pH dissociation into duners occurred at pH's lower than 10, where no ionization of tyrosines was detectable. The titration of tyrosines was independent from protein concentration.The low availability of tyrosyl residues to acetylation in deoxyhemoglobin, the cooperativity of proton binling of these residues and the change in conformation of hemoglobin concomitant with their titration are all consistent with results of Simon et al., Moffat, and Moffat et al., and with the model proposed by Perutz for explaining the heme-heme interaction. The free energy of the pK shift of the tyrosyl residues in carboxy and deoxyhemoglobin can be included in the free energy of the heme-heme interaction.     

Reactions of lignin peroxidase compounds I and II with veratryl alcohol. Transient-state kinetic characterization

Stopped-flow techniques were utilized to investigate the kinetics of the reaction of lignin peroxidase compounds I and II (LiPI and LiPII) with veratryl alcohol (VA). All rate data were collected from single turnover experiments under pseudo first-order conditions. The reaction of LiPI with VA strictly obeys second-order kinetics over the pH range 2.72-5.25 as demonstrated by linear plots of the pseudo first-order rate constants versus concentrations of VA. The second-order rate constants are strongly dependent on pH and range from 2.62 x 10(6) M-1 s-1 (pH 2.72) to 1.45 x 10(4) M-1 s-1 (pH 5.25). The reaction of LiPII and VA exhibits saturation behavior when the observed pseudo first-order rate constants are plotted against VA concentrations. The saturation phenomenon is quantitatively explained by the formation of a 1:1 LiPII-substrate complex. Results of kinetic and rapid scan spectral analyses exclude the formation of a LiPII-VA cation radical complex. The first-order dissociation rate constant and the equilibrium dissociation constant for the LiPII reaction are also pH dependent. Binding of VA to LiPII is controlled by a heme-linked ionizable group of pKa approximately 4.2. The pH profiles of the second-order rate constants for the LiPI reaction and of the first-order dissociation constants for the LiPII reaction both demonstrate two pKa values at approximately 3.0 and approximately 4.2. Protonated oxidized enzyme intermediates are most active, suggesting that only electron transfer, not proton uptake from the reducing substrate, occurs at the enzyme active site. These results are consistent with the one-electron oxidation of VA to an aryl cation radical by LiPI and LiPII.     

The anomalous pKa of Tyr-9 in glutathione S-transferase A1-1 catalyzes product release

The pKa of the catalytic Tyr-9 in glutathione S-transferase (GST) A1-1 is lowered from 10.3 to approximately 8.1 in the apoenzyme and approximately 9.0 with a GSH conjugate bound at the active site. However, a clear functional role for the unusual Tyr-9 pKa has not been elucidated. GSTA1-1 also includes a dynamic C terminus that undergoes a ligand-dependent disorder-to-order transition. Previous studies suggest a functional link between Tyr-9 ionization and C-terminal dynamics. Here we directly probe the role of Tyr-9 ionization in ligand binding and C-terminal conformation. An engineered mutant of rGSTA1-1, W21F/F222W, which contains a single Trp at the C terminus, was used as a fluorescent reporter of pH-dependent C-terminal dynamics. This mutant exhibited a pH-dependent change in Trp-222 emission properties consistent with changes in C-terminal solvation or conformation. The apparent pKa values for the conformational transition were 7.9 +/- 0.1 and 9.3 +/- 0.1 for the apoenzyme and ligand-bound enzyme, respectively, in excellent agreement with the pKa for Tyr-9 in these states. The Y9F/W21F/F222W mutant, however, exhibited no such pH-dependent changes. Time-resolved fluorescence anisotropy studies revealed a ligand-dependent, Tyr-9-dependent, change in the order parameter of Trp-222. However, no pH dependence was observed. In equilibrium and pre-steady-state ligand binding studies, product conjugate had a decreased equilibrium binding affinity (KD), concomitant with increased binding and dissociation rates, at higher pH values. Furthermore, the recovered pKa values for the pH-dependent microscopic rate constants ranged from 7.7 to 8.4, also in agreement with the pKa of Tyr-9. In contrast, the Y9F/W21F/F222W mutant had no pH-dependent transition in KD or rate constants for ligand binding or dissociation. The combined results indicate that the macroscopic populations of "open" and "closed" states of the C terminus are not determined solely by the ionization state of Tyr-9. However, the rates of transition between these states are faster for the ionized Tyr-9. The ionized Tyr-9 states provide a parallel pathway for product dissociation, which is kinetically and thermodynamically favored. In silico kinetic models further support the functional role for the parallel dissociation pathway provided by ionized Tyr-9.     

Relaxation spectra of yeast hexokinases. Isomerization of the enzyme.

Yeast hexokinase isozymes P1 and P11 exhibit a pH dependent, rapid relaxation process at 15 degrees C at enzyme concentrations of 100-474 muM and over a pH range of 6-8. The process was detected by equilibrium temperature jump spectroscopy using the indicator probe phenol red. The value of 1/tau varies from about 6 ms-1 at pH 8 for both isozymes to 50 ms-1 for P1 and 85 ms-1 for P11 at pH 6. The data are consistent with a mechanism involving an enzyme isomerization coupled to an ionization. The forward rate constant for the isomerization of the proposed mechanism varies between 3 and 7 ms-1; the ratio of the reverse rate constant to the ionization Ka is between 0.5 and 2 X 10(11) M-1 S-1; the estimated pKa varies between 5.5 and 6.1. The ranges of values in rate constants and pKa represent variations observed between preparations of the same isozyme and between isozymes. The isomerization rate is at least 50 times faster than catalysis under all conditions and the pKa is lower than that controlling activity. The rate of isomerization is unchanged by addition of sugar and nucleotide ligands, but the amplitude of the process is perturbed. These data imply that isomerizing and ionizing forms are sensitive to events at the active site. These equilibria between forms of hexokinase are fast enough, and have the right properties, to be important to the mechanism and regulation of the enzyme.     

pH-dependent conformational states of horse liver alcohol dehydrogenase.

The quenching of liver alcohol dehydrogenase protein fluorescence at alkaline pH indicates two conformational states of the enzyme with a pKa of 9.8+/-0.2, shifted to 10.6+/-0.2 in D2O. NAD+ and 2-p-toluidinonaphthalene-6-sulfonate, a fluorescent probe competitive with coenzyme, bind to the acid conformation of the enzyme. The pKa of the protein-fluorescence quenching curve is shifted toward 7.6 in the presence of NAD+, and the ternary complex formation with NAD+ and trifluoroethanol results in a pH-independent maximal quench. At pH (pD) 10.5, the rate constant for NAD+ binding was 2.6 times faster in D2O2 than in H2O due to the shift of the pKa. Based on these results, a scheme has been proposed in which the state of protonation of an enzyme functional group with a pKa of 9.8 controls the conformational state of the enzyme. NAD+ binds to the acid conformation and subsequently causes another conformational change resulting in the perturbation of the pKa to 7.6. Alcohol then binds to the unprotonated form of the functional group with a pKa of 7.6 in the binary enzyme-NAD+ complex and converts the enzyme to the alkaline conformation. Thus, at neutral pH liver alcohol dehydrogenase undergoes two conformational changes en route to the ternary complex in which hydride transfer occurs.     

Proton nuclear magnetic resonance studies on the iodide binding by horseradish peroxidase

Binding of an iodide ion to horseradish peroxidase was studied by following the hyperfine-shifted proton nuclear magnetic resonance signals of the enzyme. For the enzyme in an iodide-free solution, the spectra of hyperfine-shifted methyl region were only slightly affected by varying pH. In the presence of iodide (200 mM), however, both chemical shifts and line widths of the heme peripheral 1- and 8-methyl proton signals were markedly affected by the pH change from 7 to 4 and broadened at pH 4. From the change in peak heights of these signals at various concentrations of iodide, the dissociation constant of the iodide to the enzyme was calculated to be about 100 mM at pH 4.0. The peak derived from the proximal histidyl imidazole N epsilon-H proton was not perturbed by the addition of 200 mM iodide at pH 4.0 and 7.1. The rate of oxidation of iodide with hydrogen peroxide catalyzed by the enzyme was increased with decreasing pH, indicating the participation of an ionizable group with the pKa value of 4.0. Optical difference spectrum studies showed that iodide exerts no effect both at pH 4.0 and 7.4 on the binding affinity of resorcinol which is associated with the enzyme in the vicinity of the heme peripheral 8-CH3 group. These results suggest that an iodide ion binds to the enzyme at almost equal distance from the heme peripheral 1- and 8-methyl groups at the distal side of the heme and that the interaction becomes stronger in acidic medium with protonation of the ionizable group with the pKa value of 4.0.     

Catalytic activity of Ntau-carboxymethylhistidine-12 ribonuclease: pH dependence.

The pH-dependence of RNAase A and of Ntau-carboxymethylhistidine-12-RNAase (ribonucleate 3'-pyrimidino-oligonucleotidohydrolase) catalysis was studied. Apparent acid dissociation constants were obtained by least squares analysis of the kinetics data. These dissociation constants were compared with pKa values of model imidazole compounds, and with pKa values of histidine residues 12 and 119 on the protein. The shapes of the kcat versus pH profiles for RNAase A and its carboxymethyl derivative are very similar, from which it is concluded that the mechanism of catalysis is closely similar in the two proteins. Apparent pKa values obtained from the kinetic data are higher for the carboxymethylated protein than for RNAase A, as are the pKa values of residues 12 and 119. The similar shifts are consistent with the conclusions that both these residues are functionally significant in native and modified enzyme, and that an unblocked tau-nitrogen on histidine-12 is not essential for activity. From the enzyme's catalytic dependence on pH, and the NMR determined pKa values we propose that histidine 12 and 119 function catalytically in their basic and acidic forms respectively.