Alteration of the specificity of malate dehydrogenase by chemical modulation of an active site arginine

Malate dehydrogenase from Escherichia coli is highly specific for the oxidation of malate to oxaloacetate. The technique of site-specific modulation has been used to alter the substrate binding site of this enzyme. Introduction of a cysteine in place of the active site binding residue arginine 153 results in a mutant enzyme with diminished catalytic activity, but with K(m) values for malate and oxaloacetate that are surprisingly unaffected. Reaction of this introduced cysteine with a series of amino acid analog reagents leads to the incorporation of a range of functional groups at the active site of malate dehydrogenase. The introduction of a positively charged group such as an amine or an amidine at this position results in improved affinity for several inhibitors over that observed with the native enzyme. However, the recovery of catalytic activity is less dramatic, with less than one third of the native activity achieved with the optimal reagents. These modified enzymes do have altered substrate specificity, with alpha-ketoglutarate and hydroxypyruvate no longer functioning as alternative substrates.     

pH-dependent changes of intrinsic fluorescence of chemically modified liver alcohol dehydrogenases.

Horse liver alcohol dehydrogenase specifically carboxymethylated on cysteine-46 (a ligand to the zinc in the active site) or acetimidylated on 25 of the 30 lysine residues per subunit (including residue 228) was studied. The tryptophan fluorescence of these enzymes decreased by 35% as pH was increased, with an apparent pKa of 9.8 +/- 0.2, identical with that of native enzyme. Native enzyme in the presence of 30mM-imidazole, which displaces a water molecule ligated to the zinc, also had a pKa of 9.8. The ionoizable group is thus neither the water molecule nor one of the modified groups. Binding of NAD+ shifted the pKa for the fluorescence transition to 7.6 with native enzyme and to 9.0 with acetimidylated enzyme, but did not shift the pKa of carboxymethylated enzyme. Binding of NAD+ and trifluoroethanol, an unreactive alcohol, gave maximal fluorescence quenching at pH7 with all three enzymes. The acetimidylated enzyme--NAD+--trifluoroethanol complex had an apparent pKa of 5.0, but the pK of the native enzyme complex was experimentally inaccessible. The results are interpreted in terms of coupled equilibria between two different conformational states. On binding of NAD+, the modified enzymes apparently change conformation less readily than does native enzyme, but binding of alcohol can drive the change to completion.     

pK values for active site residues of carboxypeptidase A

The phenolic group of active site residue Tyr-248 in carboxypeptidase A has a pKa value of 10.06, as determined from the pH dependence of its rate of nitration by tetranitromethane. The decrease in enzyme activity (kcat/Km) in alkaline solution, characterized by a pKa value of approximately 9.0 (for cobalt carboxypeptidase A), is associated with the protonation state of an imidazole ligand of the active-site metal ion, as indicated by a selective pH dependence of the 1H NMR spectrum of the enzyme. Inhibition of the cobalt-substituted enzyme by 2-(1-carboxy-2-phenylethyl)phenol and its 4,6-dichloro- and 4-phenylazo-derivatives confirms that the decrease in enzyme activity (kcat/Km) in acidic solution, characterized by a pKa value of 5.8, is due to the protonation state of a water molecule bound to the active-site metal ion in the absence of substrate. Changes in the coordination number of the active-site metal ion are seen in its visible absorption spectrum as a consequence of binding of the phenolic inhibitors. Conventional concepts regarding the mechanisms of the enzyme are brought into question.     

The involvement of histidine at the active site of Harding-Passey mouse melanoma tyrosinase

The nature of the essential residues at the active site of Harding-Passey mouse melanoma tyrosinase has been explored by kinetic and photochemical modification studies. Km for L-dopa depends strongly on pH, so that acidic pH prevents the formation of the enzyme-substrate complex because the protonation of an enzyme group with a pKa of 6.6. Halide ions inhibit competitively the enzyme activity, being F the more potent one. This inhibition is also pH-dependent, showing the involvement of a protonatable group of the enzyme with apparent pKa ranging from 5.9 to 7.0. Tyrosinase has also been modified with visible light using Rose Bengal as photosensitizer, yielding a pH-dependent photoinactivation, characteristic of histidyl residues. All these results strongly support that histidine plays an important role in the dopa-oxidase activity of the enzyme, very probably acting as the ligand of copper at the active site of the enzyme.     

Characterization of recombinant rat cathepsin B and nonglycosylated mutants expressed in yeast. New insights into the pH dependence of cathepsin B-catalyzed hydrolyses.

The cysteine proteinase rat cathepsin B was expressed in yeast in an active form and was found to be heterogeneously glycosylated at the consensus sequence for N-linked oligosaccharide substitution. Purified enzyme fractions containing the highest levels of glycosylation were shown to have reduced activity. A glycosylation minus mutant constructed by site-directed mutagenesis (by changing the Ser to Ala in the consensus sequence) was still secreted by the yeast and was shown to be functionally identical with purified rat liver cathepsin B. Recombinant cathepsin B was used to further characterize the pH dependence of cathepsin B-catalyzed hydrolyses using 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA) substrates with arginine as the P1, and either arginine or phenylalanine as the P2 residue. The AMC and pNA groups give insights into the leaving group binding site (P') of cathepsin B. These studies show for the first time that at least seven dissociable groups are involved in substrate binding and hydrolysis in cathepsin B activity. Two of these groups, with pKa values of 6.9 and 7.7 in the recombinant enzyme, are in the leaving group binding site and are most likely His110 and His111. The same groups in rat liver cathepsin B have higher pKa values than in recombinant cathepsin B, but have identical function in the two enzymes. Two other groups are probably the active site Cys29 and His199 with pKa values of 3.6 and 8.6, respectively. A group with a pKa of 5.1 interacts with substrates containing Arg at P2, and the group is most likely Glu245. The remaining two groups, one with a pKa of about 4.9 and the other about 5.3, are most likely carboxyl residues possibly interacting with Arg at P1 in the substrate. The possible candidates on the basis of the x-ray structure are Asp22, Asp69, Glu171, and Glu122, all found within a 13 A radius from the active site thiol of Cys29.     

Catalytic mechanism of the dihydrofolate reductase reaction as determined by pH studies

The variation with pH of the kinetic parameters of the reaction catalyzed by dihydrofolate reductase from Escherichia coli has been determined with the aim of elucidating the chemical mechanism of the reaction. The (V/K)DHF and V profiles indicated that protonation enhances the observed rate of interaction of dihydrofolate (DHF) with the enzyme-NADPH complex as well as the maximum velocity of the reaction. The pKa value of 8.09 observed in the (V/K)DHF profile is similar to that of 7.9 observed in the Ki profile for 2,4-diamino-6,7-dimethylpteridine while the pKa value of the V profile is displaced to 8.4. From the magnitude of the pH-independent value for (V/K)DHF, it is concluded that unprotonated dihydrofolate must react, at neutral pH, with the protonated form of the enzyme. The D(V/K)DHF value is independent of pH and equal to unity whereas the DV value varies as a wave function of pH with limiting values of 1.5 and 1.0 at low and high pH, respectively. It is proposed that dihydrofolate reacts with the unprotonated enzyme-NADPH complex to form a dead-end complex and with the protonated form of the same complex to form a productive complex. Further, it is considered that the protonated carboxyl of Asp-27 at the active site of the enzyme is responsible for the protonation of the N-5 nitrogen of dihydrofolate and that this protonation precedes and facilitates hydride transfer.     

Tyrosine 8 contributes to catalysis but is not required for activity of rat liver glutathione S-transferase, 1-1.

Reaction of rat liver glutathione S-transferase, isozyme 1-1, with 4-(fluorosulfonyl)benzoic acid (4-FSB), a xenobiotic substrate analogue, results in a time-dependent inactivation of the enzyme to a final value of 35% of its original activity when assayed at pH 6.5 with 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. The rate of inactivation exhibits a nonlinear dependence on the concentration of 4-FSB from 0.25 mM to 9 mM, characterized by a KI of 0.78 mM and kmax of 0.011 min-1. S-Hexylglutathione or the xenobiotic substrate analogue, 2,4-dinitrophenol, protects against inactivation of the enzyme by 4-FSB, whereas S-methylglutathione has little effect on the reaction. These experiments indicate that reaction occurs within the active site of the enzyme, probably in the binding site of the xenobiotic substrate, close to the glutathione binding site. Incorporation of [3,5-3H]-4-FSB into the enzyme in the absence and presence of S-hexylglutathione suggests that modification of one residue is responsible for the partial loss of enzyme activity. Tyr 8 and Cys 17 are shown to be the reaction targets of 4-FSB, but only Tyr 8 is protected against 4-FSB by S-hexylglutathione. DTT regenerates cysteine from the reaction product of cysteine and 4-FSB, but does not reactivate the enzyme. These results show that modification of Tyr 8 by 4-FSB causes the partial inactivation of the enzyme. The Michaelis constants for various substrates are not changed by the modification of the enzyme. The pH dependence of the enzyme-catalyzed reaction of glutathione with CDNB for the modified enzyme, as compared with the native enzyme, reveals an increase of about 0.9 in the apparent pKa, which has been interpreted as representing the ionization of enzyme-bound glutathione; however, this pKa of about 7.4 for modified enzyme remains far below the pK of 9.1 for the -SH of free glutathione. Previously, it was considered that Tyr 8 was essential for GST catalysis. In contrast, we conclude that Tyr 8 facilitates the ionization of the thiol group of glutathione bound to glutathione S-transferase, but is not required for enzyme activity.     

Reactivation of human placental 17 beta,20 alpha-hydroxysteroid dehydrogenase affinity alkylated by estrone 3-(bromoacetate): topographic studies with 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether)

Estradiol 17 beta-dehydrogenase and 20 alpha-hydroxysteroid dehydrogenase, oxidoreductase activities copurified from the cytosol of human-term placenta as a homogeneous protein (native enzyme), were reactivated at equal rates to 100% activity following complete inactivation in the presence of cofactor (NADPH) with the affinity alkylator estrone 3-(bromoacetate). Reactivation was accomplished by base-catalyzed hydrolysis of steroidal ester-amino acid linkages in the enzyme active site. The rate of enzyme reactivation was pH dependent. In identical studies without NADPH, only 12% of the original enzyme activity was restored. Completely reactivated enzyme was repurified by dialysis. Enzyme in control mixtures (control enzyme) that contained estrone in place of alkylator was treated the same as the reactivated enzyme. Reactivated enzyme exhibited a 6.0-fold lower affinity for common substrates, a 1.8-fold lesser affinity for NAD+ and NADH, and the same affinity for NADP+ and NADPH compared to control enzyme. In incubations that included NADPH, the reactivated enzyme maintained full activity during a 20-h second exposure to estrone 3-(bromoacetate), but in identical incubations without NADPH, the reactivated enzyme was rapidly inactivated at the same rate as the control and native enzymes. The control and reactivated enzymes were inactivated at equal rates by 16 alpha-(bromoacetoxy)estradiol 3-(methyl ether) in the presence or absence of cofactor (NADP+) and exhibited similar Kitz and Wilson inhibition constants for this affinity alkylator. Estrone 3-(bromo[2'-14C]acetate) incubated with native enzyme and NADPH produced radiolabeled 3-(carboxymethyl)histidine and S-(carboxymethyl)cysteine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

The reaction of trimethylamine dehydrogenase with trimethylamine

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.     

Brain succinic semialdehyde dehydrogenase. Reactions of sulfhydryl residues connected with catalytic activity.

Incubation of an NAD+-dependent succinic semialdehyde dehydrogenase from bovine brain with 4-dimethylaminoazobenzene-4-iodoacetamide (DABIA) resulted in a time-dependent loss of enzymatic activity. This inactivation followed pseudo first-order kinetics with a second-order rate constant of 168 m(-1).min(-1). The spectrum of DABIA-labeled enzyme showed a characteristic peak of the DABIA alkylated sulfhydryl group chromophore at 436 nm, which was absent from the spectrum of the native enzyme. A linear relationship was observed between DABIA binding and the loss of enzyme activity, which extrapolates to a stoichiometry of 8.0 mol DABIA derivatives per mol enzyme tetramer. This inactivation was prevented by preincubating the enzyme with substrate, succinic semialdehyde, but not by preincubating with coenzyme NAD+. After tryptic digestion of the enzyme modified with DABIA, two peptides absorbing at 436 nm were isolated by reverse-phase HPLC. The amino acid sequences of the DABIA-labeled peptides were VCSNQFLVQR and EVGEAICTDPLVSK, respectively. These sites are identical to the putative active site sequences of other brain succinic semialdehyde dehydrogenases. These results suggest that the catalytic function of succinic semialdehyde dehydrogenase is inhibited by the specific binding of DABIA to a cysteine residue at or near its active site.     

Implications for an ionized alkyl-enzyme intermediate during StEH1-catalyzed trans-stilbene oxide hydrolysis

The catalytic mechanism of epoxide hydrolase (EC 3.3.2.3) involves acid-assisted ring opening of the oxirane during the alkylation half-reaction of hydrolysis. Two tyrosyl residues in the active site of epoxide hydrolases have been shown to contribute to the catalysis of enzyme alkylation, but their mechanism of action has not been fully described. We have investigated the involvement of the active site Tyr154 and Tyr235 during S,S-trans-stilbene oxide hydrolysis catalyzed by potato epoxide hydrolase StEH1. Tyr phenol ionizations of unliganded enzyme as well as under pre-steady-state conditions during catalysis were studied by direct absorption spectroscopy. A transient UV absorption, indicative of tyrosinate formation, was detected during the lifetime of the alkyl-enzyme intermediate. The apparent pKa of Tyr ionization was 7.3, a value more than 3 pH units below the estimated pKa of protein Tyr residues in the unliganded enzyme. In addition, the pH dependencies of microscopic kinetic rates of catalyzed S,S-trans-stilbene oxide hydrolysis were determined. The alkylation rate increased with pH and displayed a pKa value identical to that of Tyr ionization (7.3), whereas the reverse (epoxidation) reaction did not display any pH dependence. The rate of alkyl-enzyme hydrolysis was inversely dependent on tyrosinate formation, decreasing with its buildup in the active site. Since alkyl-enzyme hydrolysis is the rate-limiting step of the overall reaction, kcat displayed the same decrease with pH as the hydrolysis rate. The compiled results suggested that the role of the Tyr154/Tyr235 pair was not as ultimate proton donor to the alkoxide anion but to stabilize the negatively charged alkyl-enzyme through electrophilic catalysis via hydrogen bonding.     

Use of a site-directed triple mutant to trap intermediates: demonstration that the flavin C(4a)-thiol adduct and reduced flavin are kinetically competent intermediates in mercuric ion reductase

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala135Cys140Ala558Ala559), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH- form accumulates. The second intermediate is a flavin C(4a)-Cys140 thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP+. The conversion of the Cys135-Cys140 disulfide in wild-type enzyme to the monothiol Cys140 in ACAA and the elevated pKa of Cys140 (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH- and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH- to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH2 form in the reductive half-reaction for this class of enzymes.     

Active site of mercuric reductase resides at the subunit interface and requires Cys135 and Cys140 from one subunit and Cys558 and Cys559 from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation

Mercuric reductase catalyzes the two-electron reduction of Hg(II) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(II) by bacteria. The enzyme is an alpha 2 homodimer and possesses two pairs of cysteine residues, Cys135 and Cys140 (redox-active pair) and Cys558 and Cys559 (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala135Ala140Cys558Cys559 (AACC) and Cys135Cys140Ala558Ala559 (CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an HgR phenotype, some 10(4)-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.     

Effects of two ionizing groups on the active site of human carbonic anhydrase B.

Investigation of some pH-dependent properties of human erythrocyte carbonic anhydrase B indicate that the active site is influenced by at least two charged groups. The properties studied include the pH dependence of inhibition of native, monocarboxamidomethyl, and monocarboxymethyl enzymes by iodide ion and the pH dependence of the visible spectra of the cobalt derivatives of these enzymes. One ionizing group has a pKa of about 7.3 in the native enzyme, 8.2 in the carboxyamidomethyl enzyme, and 9.0 in the carboxymethyl enzyme. It has a major influence on activity and anion inhibition, and on the visible spectra of the cobalt enzymes. A second group has a pKa of about 6.1 in native and modified enzymes. When zinc is at the active site, the secondary group in its acidic form decreases the Ki for I-. With the carboxyamidomethyl and carboxymethyl enzymes, the Ki decreases by about an order of magnitude. However, if cobalt is substituted for zinc in the modified enzymes, this group does not influence the Ki for I- and the binding of I- does not influence the pKa of the spectral transitions caused by ionization of this secondary group. In the case of nonalkylated Co2+-enzyme, another ionizing group with a pK of about 6.2 prevents the binging of I- at low pH. These results show that the active site is altered when cobalt is substituted for zinc in carbonic anhydrase B.     

Conformation, stability, and active-site cysteine titrations of Escherichia coli D26A thioredoxin probed by Raman spectroscopy.

The active-site cysteines (Cys 32 and Cys 35) of Escherichia coli thioredoxin are oxidized to a disulfide bridge when the protein mediates substrate reduction. In reduced thioredoxin, Cys 32 and Cys 35 are characterized by abnormally low pKa values. A conserved side chain, Asp 26, which is sterically accessible to the active site, is also essential to oxidoreductase activity. pKa values governing cysteine thiol-thiolate equilibria in the mutant thioredoxin, D26A, have been determined by direct Raman spectrophotometric measurement of sulfhydryl ionizations. The results indicate that, in D26A thioredoxin, both sulfhydryls titrate with apparent pKa values of 7.5+/-0.2, close to values measured previously for wild-type thioredoxin. Sulfhydryl Raman markers of D26A and wild-type thioredoxin also exhibit similar band shapes, consistent with minimal differences in respective cysteine side-chain conformations and sulfhydryl interactions. The results imply that neither the Cys 32 nor Cys 35 SH donor is hydrogen bonded directly to Asp 26 in the wild-type protein. Additionally, the thioredoxin main-chain conformation is largely conserved with D26A mutation. Conversely, the mutation perturbs Raman bands diagnostic of tryptophan (Trp 28 and Trp 31) orientations and leads to differences in their pH dependencies, implying local conformational differences near the active site. We conclude that, although the carboxyl side chain of Asp 26 neither interacts directly with active-site cysteines nor is responsible for their abnormally low pKa values, the aspartate side chain may play a role in determining the conformation of the enzyme active site.     

Role of the reactive cysteine residue in restriction endonuclease Cfr9I.

Chemical modification studies were performed to elucidate the role of Cys-residues in the catalysis/binding of restriction endonuclease Cfr9I. Incubation of restriction endonuclease Cfr9I with N-ethylmaleimide (NEM), iodoacetate, 5,5'-dithiobis (2-nitrobenzoic acid) at pH 7.5 led to a complete loss of the catalytic activity. However, no enzyme inactivation was detectable after modification of the enzyme with iodoacetamide and methyl methanethiosulfonate. Complete protection of the enzyme against inactivation by NEM was observed in the presence of substrate implying that Cys-residues may be located at or in the vicinity of the active site of enzyme. Direct substrate-binding studies of native and modified restriction endonuclease Cfr9I using a gel-mobility shift assay indicated that the modification of the enzyme by NEM was hindered by substrate binding. A single Cys-residue was modified during the titration of the enzyme with DTNB with concomitant loss of the catalytic activity. The pH-dependence of inactivation of Cfr9I by NEM revealed the modification of the residue with the pKa value of 8.9 +/- 0.2. The dependence of the reaction rate of substrate hydrolysis by Cfr9I versus pH revealed two essential residues with pKa values of 6.3 +/- 0.15 and 8.7 +/- 0.15, respectively. The evidence presented suggests that the restriction endonuclease Cfr9I contains a reactive sulfhydryl residue which is non-essential for catalysis, but is located at or near the substrate binding site.     

pH dependence of the kinetic parameters of L-asparaginase.

The concentration dependence of the rate of hydrolysis of L-asparagine by Escherichia coli L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1) has been measured over the range pH 4.5 to pH 9.1 by a direct spectrophotometric assay at 220 nm and by a coupled assay utilizing glutamate dehydrogenase to detect the ammonia produced. The velocity of the hydrolysis reaction at saturating levels of substrate is independent of pH over this interval. The plot of V/km over the same interval is bell-shaped, being dependent on pKa values of 6.58 and 8.69. The higher pKa is attributed to the amino group of asparagine. The lower pKa is associated with the enzyme active site and is probably due to an imidazole group.     

Effect of Asp-235-->Asn substitution on the absorption spectrum and hydrogen peroxide reactivity of cytochrome c peroxidase.

The spectroscopic properties of a mutant cytochrome c peroxidase, in which Asp-235 has been replaced by an asparagine residue, were examined in both nitrate and phosphate buffers between pH 4 and 10.5. The spin state of the enzyme is pH dependent, and four distinct spectroscopic species are observed in each buffer system: a predominantly high-spin Fe(III) species at pH 4, two distinct low-spin forms between pH 5 and 9, and the denatured enzyme above pH 9.3. The spectrum of the mutant enzyme at pH 4 is dependent upon specific ion effects. Increasing the pH above 5 converts the mutant enzyme to a predominantly low-spin hydroxy complex. Subsequent conversion to a second low-spin form is essentially complete at pH 7.5. The second low-spin form has the distal histidine, His-52, coordinated to the heme iron. To evaluate the effect of the changes in coordination state upon the reactivity of the enzyme, the reaction between hydrogen peroxide and the mutant enzyme was also examined as a function of pH. The reaction of CcP(MI,D235N) with peroxide is biphasic. At pH 6, the rapid phase of the reaction can be attributed to the bimolecular reaction between hydrogen peroxide and the hydroxy-ligated form of the mutant enzyme. Despite the hexacoordination of the heme iron in this form, the bimolecular rate constant is approximately 22% that of pentacoordinate wild-type yeast cytochrome c peroxidase. The bimolecular reaction of the mutant enzyme with peroxide exhibits the same pH dependence in nitrate-containing buffers that has been described for the wild-type enzyme, indicating a loss of reactivity with the protonation of a group with an apparent pKa of 5.4. This observation eliminates Asp-235 as the source for this heme-linked ionization and strengthens the hypothesis that the pKa of 5.4 is associated with His-52. The slower phase of the reaction between peroxide and the mutant enzyme saturates at high peroxide concentration and is attributed to conversion of unreactive to reactive forms of the enzyme. The fraction of enzyme which reacts via the slow phase is dependent upon both pH and specific ion effects.     

The role of an essential histidine residue of yeast alcohol dehydrogenase.

1. Inactivation of yeast alcohol dehydrogenase for diethyl pyrocarbonate indicates that one histidine residue per enzyme subunit is necessary for enzymic activity. The inactivated enzyme regains its activity over a period of days. 2. Enzyme modified by diethyl pyrocarbonate can form the binary enzyme - NADH complex with the same maximum NADH-binding capacity as that of native enzyme. Modified enzyme cannot form normal ternary complexes of the type enzyme - NADH - acetamide and enzyme - NAD+ - pyrazole, which are characteristic of native enzyme. 3. The rate constant for the reaction of enzyme with diethyl pyrocarbonate has been determined over the pH range 5.5--9. The histidine residue involved has approximately the same pKa as free histidine, but is 10-fold more reactive than free histidine.