Kinetic and spectroscopic studies of Tritrichomonas foetus low-molecular weight phosphotyrosyl phosphatase. Hydrogen bond networks and electrostatic effects

Virtually all of the eukaryotic low-molecular weight protein tyrosine phosphatases (LMW PTPases) studied to date contain a conserved, high-pK(a) histidine residue that is hydrogen bonded to a conserved active site asparagine residue of the phosphate binding loop. However, in the putative enzyme encoded by the genome of the trichomonad parasite Tritrichomonas foetus, this otherwise highly conserved histidine is replaced with a glutamine residue. We have cloned the gene, expressed the enzyme, demonstrated its catalytic activity, and examined the structural and functional roles of the glutamine residue using site-directed mutagenesis, kinetic measurements, and NMR spectroscopy. Titration studies of the two native histidine residues in the T. foetus enzyme as monitored by (1)H NMR revealed that H44 has a pK(a) of 6.4 and H143 has a pK(a) of 5.3. When a histidine residue was introduced in place of the native glutamine at position 67, a pK(a) of 8.2 was measured for this residue. Steady state kinetic methods were employed to study how mutation of the native glutamine to alanine, asparagine, and histidine affected the catalytic activity of the enzyme. Examination of k(cat)/K(m) showed that Q67H exhibits a substrate selectivity comparable to that of the wild-type (WT) enzyme, while Q67N and Q67A show reduced activity. The effect of pH on the reaction rate was examined. Importantly, the pH-rate profile of the WT TPTP enzyme revealed a much more clearly defined acidic limb than that which can be observed for other wild-type LMW PTPases. The pH-rate curve of the Q67H mutant shows a shift to a lower pH optimum relative to that seen for the wild-type enzyme. The Q67N and Q67A mutants showed curves that were shifted to higher pH optima. Although the active site of this enzyme is likely to be similar to that of other LMW PTPases, the hydrogen bonding and electrostatic changes afford new insight into factors affecting the pH dependence and catalysis by this family of enzymes.     

Dual-specific Cdc25B phosphatase: in search of the catalytic acid

Cdc25 is a dual-specificity phosphatase that catalyzes the activation of the cyclin-dependent kinases, thus causing initiation and progression of successive phases of the cell cycle. Although it is not significantly structurally homologous to other well-characterized members, Cdc25 belongs to the class of well-studied cysteine phosphatases as it contains their active site signature motif. However, the catalytic acid needed for protonation of the leaving group has yet to be identified. To elucidate the role and identity of this key catalytic residue, we have performed a detailed pH-dependent kinetic analysis of Cdc25B. The pK(a) of the catalytic cysteine was found to be 5.6-6.3 in steady state and one-turnover burst experiments using the small molecule substrates p-nitrophenyl phosphate and 3-O-methylfluorescein phosphate. Interestingly, Cdc25B does not exhibit the typical bell-shaped pH-rate profile with small molecule substrates seen in other cysteine phosphatases and indicative of the catalytic acid because it lacks pH dependence between 6.5 and 9. Reactions of Cdc25B with the natural substrate Cdk2-pTpY/CycA, however, did yield a bell-shaped pH-rate profile with a pK(a) of 6.1 for the catalytic acid residue. Recent structural studies of Cdc25 have suggested that Glu474 [Fauman, E. B., et al. (1998) Cell 93, 617-625] or Glu478 [Reynolds, R. A., et al. (1999) J. Mol. Biol. 293, 559-568] could function as the catalytic acid in Cdc25B. Using site-directed mutagenesis and truncation experiments, however, we found that neither of these residues, nor the unstructured C-terminus, is responsible for the observed pH dependence. These results indicate that the catalytic acid does not appear to lie within the known structure of Cdc25B and may lie on its protein substrate.     

Lysine-73 is involved in the acylation and deacylation of beta-lactamase

Lysine 73 is a conserved active-site residue in the class A beta-lactamases, as well as other members of the serine penicillin-sensitive enzyme family; its role in catalysis remains controversial and uncertain. Mutation of Lys73 to alanine in the beta-lactamase from Bacillus licheniformis resulted in a substantial reduction in both turnover rate (k(cat)) and catalytic efficiency (k(cat)/K(m)), and a very significant shift in pK(1) to higher pH in the bell-shaped pH-rate profiles (k(cat)/K(m)) for several penicillin and cephalosporin substrates. The increase in pK(1) is consistent with the removal of the positive ammonium group of the lysine from the proximity of Glu166, to which the acid limb has been ascribed. The alkaline limb of the k(cat)/K(m) vs profiles is not shifted appreciably, as might have been expected if this limb reflected the ionization of Lys73 in the wild-type enzyme. The k(cat)/K(m) at the pH optimum for the mutant was down about 200-fold for penicillins and around 10(4) for cephalosporins, compared to the wild-type, suggesting significant differences in the mechanisms for catalysis of penicillins compared to cephalosporins. Burst kinetics were observed with several substrates assayed with K73A beta-lactamase, indicating an underlying branched-pathway kinetic scheme, and rate-limiting deacylation. FTIR analysis was used to determine whether acylation or deacylation was rate-limiting. In general, acylation was the rate-limiting step for cephalosporin substrates, whereas deacylation was rate-limiting for penicillin substrates. The results indicate that Lys73 plays an important role in both the acylation and deacylation steps of the catalytic mechanism. The effects of this mutation (K73A) indicate that Lys73 does not function as a general base in the catalytic mechanism of beta-lactamase. The existence of bell-shaped pH-rate profiles for the K73A variant suggests that Lys73 is not directly responsible for either limb in such plots. It is likely that both Glu166 and Lys73 are important to each other in terms of maintaining the optimum electrostatic environment for fully efficient catalytic activity to occur.     

A catalytic triad is responsible for acid-base chemistry in the Ascaris suum NAD-malic enzyme

The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K(malate) for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K(malate) for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K(malate) pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K(malate) indicates the mutant is only active above pH 9. The data suggest that the pK present at about pH 5.6 in all of the pH profiles represents D294, and during catalysis D294 accepts a proton from K199 to allow K199 to act as a general base in the reaction. The pK for the general acid in the reaction is not observed, consistent with rapid tautomerization of enolpyruvate. No other ionizable group in the active site is likely responsible for the partial activity change observed in the pH profiles, and thus the group responsible is probably remote from the active site and the effect on activity is transmitted through the protein by a conformational change.     

Proton magnetic resonance studies of carbonic anhydrase. II. Group controlling catalytic activity.

The seven resonances observed in the histidine region of the proton magnetic resonance (pmr) spectrum of human carbonic anhydrase B and reported in the preceding paper are studied in the presence of sulfonamide, azide, cyanide, and chloride inhibitors and in metal-free, cadmium substituted, cobalt substituted, and carboxymethylated forms of the enzyme. Results indicate that the two resonances that move-downfield with increasing pH and the two that do not move with pH reflect residues located at the active site. The first two resonances are assigned to the same titratable histidine whose pK value of 8.24 corresponds to that of the group controlling catalytic activity. Addition of anions or sulfonamides, removal of zinc, or substitution of cadmium for zinc at the active site, procedures known to abolish enzymatic activity, prevent titration of this residue. Partial inhibition of carbonic anhydrase by chloride slectively increases the pK value of the group controlling catalytic activity and of the histidine with pK equals 8.24. Experiments with metal-free and cadmium carbonic anhydrases and comparisons with model systems suggest that this histidine is bound to the metal ion at high pH; at low pH this complex appears to dissociate as protons compete with the metal for the imidazole group. It is proposed that ionization of the group controlling catalytic activity represents loss of the pyrrole proton of this neutral ligand when it binds to Zn(II), forming an imidazolate anion and juxtaposing a strong base and a powerful Lewis acid at the active site. When bound to zinc as an anion, this histidine can act as a general base catalyst in the hydration of carbon dioxide and be replaced as a metal ligand by an oxygen of the substrate in the course of the reaction. The histidine-metal complex is thought to exist in a strained configuration in the active enzyme so that its imidazole-metal bond is readily broken on addition of substrates or inhibitors. This model is consistent with the available data on the enzyme and is discussed in relation to alternative proposals.     

Jack bean (Canavalia ensiformis) urease. Probing acid-base groups of the active site by pH variation.

A pH-variation study of jack bean (Canavalia ensiformis) urease steady-state kinetic parameters and of the inhibition constant of boric acid, a urease competitive inhibitor, was performed using both noninhibitory organic (MES, HEPES and CHES) and inhibitory inorganic (phosphate) buffers, in an effort to elucidate the functions exercised in the catalysis by the ionizable groups of the enzyme active site. The results obtained are consistent with the requirement for three groups utilized by urease with pK(a)s equal to 5.3+/-0.2, 6.6+/-0.2 and 9.1+/-0.4. Based on the appearance of the ionization step with pK(a)=5.3 in v(max)-pH, K(M)-pH and K(i)-pH profiles, we assigned this group as participating both in the substrate binding and catalytic reaction. As shown by its presence in v(max)-pH and K(M)-pH curves, the obvious role of the group with pK(a)=9.1 is the participation in the catalytic reaction. One function of the group featuring pK(a)=6.6, which was derived from a two-maxima v(max)-pH profile obtained upon increasing phosphate buffer concentration, an effect the first time observed for urease-phosphate systems, is the substrate binding, another possible function being modulation of the active site structure controlled by the ionic strength. It is also possible that the pK(a)=6.6 is a merger of two pK(a)s close in value. The study establishes that regular bell-shaped activity-pH profiles, commonly reported for urease, entail more complex pH-dependent behavior of the urease active site ionizable groups, which could be experimentally derived using species interacting with the enzyme, in addition to changing solution pH and ionic strength.     

Physical, kinetic and spectrophotometric studies of a NAD(P)-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633

The mandelate pathway of Pseudomonas putida ATCC 12633 comprises five enzymes and catalyzes the conversion of R- and S-mandelamide to benzoic acid which subsequently enters the beta-ketoadipate pathway. Although the first four enzymes have been extensively characterized the terminal enzyme, a NAD(P)(+)-dependent benzaldehyde dehydrogenase (BADH), remains largely undescribed. Here we report that BADH is a dimer in solution, and that DTT is necessary both to maintain the activity of BADH and to prevent oligimerization of the enzyme. Site-directed mutagenesis confirms that Cys249 is the catalytic cysteine and identifies Cys140 as the cysteine likely to be involved in inter-monomer disulfide formation. BADH can utilize a range of aromatic substrates and will also operate efficiently with cyclohexanal as well as medium-chain aliphatic aldehydes. The logV and logV/K pH-rate profiles for benzaldehyde with either NAD(+) or NADP(+) as the coenzyme are both bell-shaped. The pK(a) values on the ascending limb range from 6.2 to 7.1 while those on the descending limb range from 9.6 to 9.9. A spectrophotometric approach was used to show that the pK(a) of Cys249 was 8.4, i.e., Cys249 is not responsible for the pK(a)s observed in the pH-rate profiles.     

Chemical mechanism of the adenosine cyclic 3',5'-monophosphate dependent protein kinase from pH studies

The pH dependence of kinetic parameters and inhibitor dissociation constants for the adenosine cyclic 3',5'-monophosphate dependent protein kinase reaction has been determined. Data are consistent with a mechanism in which reactants selectively bind to enzyme with the catalytic base unprotonated and an enzyme group required protonated for peptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) binding. Binding of the peptide apparently locks both of the above enzyme residues in their correct protonation state. MgATP preferentially binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/KMgATP are pH independent. The V/K for Ser-peptide is bell-shaped with pK values of 6.2 and 8.5 estimated. The pH dependence of 1/Ki for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/KSer-peptide, while the Ki for MgAMP-PCP increases from a constant value of 650 microM above pH 8 to a constant value of 4 mM below pH 5.5. The Ki for uncomplexed Mg2+ obtained from the Mg2+ dependence of V and V/KMgATP is apparently pH independent.     

pH dependence of the kinetic parameters for 3-oxo-delta 5-steroid isomerase. Substrate catalysis and inhibition by (3S)-spiro[5 alpha-androstane-3,2'-oxiran]-17-one

The pH-rate profiles for kcatobsd and (kcat/KM)obsd at 25.0 degrees C have been measured for 3-oxo-delta 5-steroid isomerase by using 5-androstene-3,17-dione (2), 5-pregnene-3,20-dione (3), and 5(10)-estrene-3,17-dione (4) as substrates. Results from the nonsticky substrate 4 suggest values for the pK of a catalytically important group on the free enzyme (pKE) of 4.57 and the pK of the same group in the enzyme-substrate complex of 4.74. For the sticky substrates 2 and 3, pKES is ca. 4.75 and 5.5, respectively. Analysis of the (kcat/KM)obsd vs. pH profile for 2 reveals that the intermediate E X S complex decomposes to products at a rate similar to its reversion to E + S. The pH-rate profile for inhibition of the isomerase by (3S)-spiro-[5 alpha-androstane-3,2'-oxiran]-17-one (7 beta) shows values for pKE of 4.75 and pKEI of 4.90. The similarity of the pH-rate profiles for isomerization of 4 and inhibition by 7 beta suggests that both reactions may be governed by the ionization state of the same carboxyl group of the enzyme.     

HDV ribozyme activity in monovalent cations

Activity of the two ribozymes from hepatitis delta virus in monovalent salts was examined and compared to activity in Mg2+. Both ribozymes self-cleaved in high concentrations of monovalent cations, and an active site cytosine was required for cleavage activity under those conditions. Cleavage rates were 30-50-fold higher for reactions in LiCl than for reactions in NaCl or NH4Cl, and a thio effect indicated that chemistry was rate-determining for cleavage of the HDV genomic ribozyme in LiCl. Still, in LiCl, there was a more than 100-fold increase in the rate when MgCl2 was included in the reaction. However, the pH-rate profiles for the reactions in LiCl with and without MgCl2 were both bell-shaped with the pH optima in the neutral range. These findings support the idea that monovalent cations can partially substitute for divalent metal ions in the HDV ribozymes, although a divalent metal ion is more effective in supporting catalysis. The absence of a dramatic change in the general shape of pH-rate profiles in LiCl, relative to the profile for reactions including Mg2+, is in contrast to earlier data for the reactions in NaCl and limits our interpretation of the specific role played by the divalent metal ion in the catalytic mechanism.     

Proton transfer dynamics of GART: the pH-dependent catalytic mechanism examined by electrostatic calculations

The enzyme glycinamide ribonucleotide transformylase (GART) catalyzes the transfer of a formyl group from formyl tetrahydrofolate (fTHF) to glycinamide ribonucleotide (GAR), a process that is pH-dependent with pK(a) of approximately 8. Experimental studies of pH-rate profiles of wild-type and site-directed mutants of GART have led to the proposal that His108, Asp144, and GAR are involved in catalysis, with His108 being an acid catalyst, while forming a salt bridge with Asp144, and GAR being a nucleophile to attack the formyl group of fTHF. This model implied a protonated histidine with pK(a) of 9.7 and a neutral GAR with pK(a) of 6.8. These proposed unusual pK(a)s have led us to investigate the electrostatic environment of the active site of GART. We have used Poisson-Boltzmann-based electrostatic methods to calculate the pK(a)s of all ionizable groups, using the crystallographic structure of a ternary complex of GART involving the pseudosubstrate 5-deaza-5,6,7,8-THF (5dTHF) and substrate GAR. Theoretical mutation and deletion analogs have been constructed to elucidate pairwise electrostatic interactions between key ionizable sites within the catalytic site. Also, a construct of a more realistic catalytic site including a reconstructed pseudocofactor with an attached formyl group, in an environment with optimal local van der Waals interactions (locally minimized) that imitates closely the catalytic reactants, has been used for pK(a) calculations. Strong electrostatic coupling among catalytic residues His108, Asp144, and substrate GAR was observed, which is extremely sensitive to the initial protonation and imidazole ring flip state of His108 and small structural changes. We show that a proton can be exchanged between GAR and His108, depending on their relative geometry and their distance to Asp144, and when the proton is attached on His108, catalysis could be possible. Using the formylated locally minimized construct of GART, a high pK(a) for His108 was calculated, indicating a protonated histidine, and a low pK(a) for GAR(NH(2)) was calculated, indicating that GAR is in neutral form. Our results are in qualitative agreement with the current mechanistic picture of the catalytic process of GART deduced from the experimental data, but they do not reproduce the absolute magnitude of the pK(a)s extracted from fits of k(cat)-pH profiles, possibly because the static time-averaged crystallographic structure does not describe adequately the dynamic nature of the catalytic site during binding and catalysis. In addition, a strong effect on the pK(a) of GAR(NH(2)) is produced by the theoretical mutations of His108Ala and Asp144Ala, which is not in agreement with the observed insensitivity of the pK(a) of GAR(NH(2)) modeled from the experimental data using similar mutations. Finally, we show that important three-way electrostatic interactions between highly conserved His137, with His108 and Asp144, are responsible for stabilizing the electrostatic microenvironment of the catalytic site. In conclusion, our data suggest that further detailed computational and experimental work is necessary.     

Kinetic study of soybean beta-amylase. The effect of pH.

1) Beta-Amylase [EC 3.2.1.2] was prepared from defatted hawk eye soybean flour. The enzyme concentration dependence of the initial velocity for the hydrolytic reaction was investigated at pH 5.4 in the range of the enzyme concentration from 6.6 x 10(-10) M to 5.0 x 10(-6) M. It was found that the initial velocity was proportional to the enzyme concentration in this range. 2) The hydrolyses of maltodextrin (DPn = 74.4) and soluble starch catalyzed by soybean beta-amylase were investigated in the pH range from 3.0 to 9.1 at 25 degrees C, and the Michaelis constant, Km, and the maximum velocity, V, for each substrate were determined at each pH. The pH-rate profile showed a bell-shaped curve, and the pH "optimum" was at 5.85. From Dixon plots of V and V/Km, the pK values were found to be 3.5 and 8.2 for the free enzyme, and 3.5 and 8.5 for the enzyme-substrate complex. The pH-rate profile in the presence of 25% methanol (v/v) was also obtained at alkaline pH. The pKe values were the same as those in the absence of methanol. Based on these results, it was estimated that the ionizable acidic group was an amino group and the basic group was a carboxyl one.     

The pH variation of steady-state kinetic parameters of site-specific Co(2+)-reconstituted liver alcohol dehydrogenase. A mechanistic probe for the assignment of metal-linked ionizations

To identify ionizations of the active site metal-bound water in horse liver alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; EC 1.1.1.1), the pH, solvent isotope, temperature, and anion dependences of the steady-state kinetic parameters kcat and kcat/KM have been evaluated under initial velocity conditions for the native and the active site-specific Co(2+)-reconstituted enzyme. In the oxidation of benzyl alcohol, a bell-shaped pattern of four prototropic equilibria was observed under conditions of saturating concentrations of NAD+. It is shown that the ionizations governing kcat (pK1 congruent to 6.7, pK2 congruent to 10.6) belong to the ternary enzyme-NAD(+)-alcohol complex, whereas the ionizations governing kcat/KM (pK1' congruent to 7.5, pK2' congruent to 8.9) belong to the binary enzyme-NAD+ complex. The ionizations pK1 and pK1' are not influenced by metal substitution and are ascribed to His-51 on the basis of experimental estimates of their associated enthalpies of ionization. On the other hand, pK2 and pK2' are significantly decreased (delta pKa congruent to 1.0) in the Co(2+)-enzyme and are attributed to the active site metal-bound water molecule. The shape of the pH profiles requires that the metal ion coordinates a neutral water molecule in the ternary enzyme-NAD(+)-alcohol complex under physiological conditions. The possible catalytic role of the water molecule within a pentacoordinate metal ion complex in the active site is discussed.     

Kinetic studies of rat kidney gamma-glutamyltranspeptidase deacylation reveal a general base-catalyzed mechanism

The enzyme gamma-glutamyltranspeptidase (GGT) is critical to cellular detoxification and leukotriene biosynthesis processes, as well as amino acid transport in kidneys. GGT has also been implicated in many important physiological disorders, including Parkinson's disease and inhibition of apoptosis. It binds glutathione as a donor substrate and initially forms a gamma-glutamyl-enzyme complex that can then react with a water molecule or an acceptor substrate (usually an amino acid or a dipeptide) to form glutamate or a product containing a new gamma-glutamyl-isopeptide bond, respectively, thus regenerating the free enzyme. Despite its important role in human physiology, the mechanisms of the reactions catalyzed by GGT are not well-known, particularly with respect to the deacylation step. We have synthesized a series of methionine amide derivatives whose alpha-ammonium groups have different pK(a) values. By using these compounds as acceptor substrates for GGT, we have constructed a Br?nsted plot and obtained a good correlation for log(k(norm)(cat,b)/K(b)) versus pK(a)(NH+) with a slope beta(nuc) of 0.84, consistent with a rate-limiting nucleophilic attack of the substrate amine on the acyl-enzyme intermediate. Isotope effect studies have shown that there is a proton in flight at the transition state, consistent with concerted deprotonation of the nucleophilic amine effected by an unidentified general base. A bell-shaped pH-rate profile has also been obtained for the deacylation step, reflecting the pK(a) values of the acceptor substrate (and/or that of a general base residue) and of a putative general acid that may be necessary for reprotonation of the active site nucleophile upon regeneration of the free enzyme. These data allow us to propose for the first time a detailed mechanism for this important step of the GGT-mediated reaction and to speculate about the origin of its acceptor substrate specificity.     

Divalent metal derivatives of the hamster dihydroorotase domain.

Dihydroorotase (DHOase, EC 3.5.2.3) is a zinc enzyme that catalyzes the reversible cyclization of N-carbamyl-L-aspartate to L-dihydroorotate in the third reaction of the de novo pathway for biosynthesis of pyrimidine nucleotides. The recombinant hamster DHOase domain from the trifunctional protein, CAD, was overexpressed in Escherichia coli and purified. The DHOase domain contained one bound zinc atom at the active site which was removed by dialysis against the chelator, pyridine-2,6-dicarboxylate, at pH 6.0. The apoenzyme was reconstituted with different divalent cations at pH 7.4. Co(II)-, Zn(II)-, Mn(II)-, and Cd(II)-substituted DHOases had enzymic activity, but replacement with Ni(2+), Cu(2+), Mg(2+), or Ca(2+) ions did not restore activity. Atomic absorption spectroscopy showed binding of one Co(II), Zn(II), Mn(II), Cd(II), Ni(II), or Cu(II) to the enzyme, while Mg(II) and Ca(II) were not bound. The maximal enzymic activities of the active, reconstituted DHOases were in the following order: Co(II) --> Zn(II) --> Mn(II) --> Cd(II). These metal substitutions had major effects upon values for V(max); effects upon the corresponding K(m) values were less pronounced. The pK(a) values of the Co(II)-, Mn(II)-, and Cd(II)-substituted enzymes derived from pH-rate profiles are similar to that of Zn(II)-DHOase, indicating that the derived pK(a) value of 6.56 obtained for Zn-DHOase is not due to ionization of an enzyme-metal aquo complex, but probably a histidine residue at the active site. The visible spectrum of Co(II)-substituted DHOase exhibits maxima at 520 and 570 nm with molar extinction coefficients of 195 and 210 M(-1) cm(-1), consistent with pentacoordination of Co(II) at the active site. The spectra at high and low pH are different, suggesting that the environment of the metal binding site is different at these pHs where the reverse and forward reactions, respectively, are favored.     

Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase/oxidase

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline, L-pipecolic acid and sarcosine, with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD, FMN and ATP per alphabeta complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline, for which a bell-shaped dependence (pK(a) values 7.0 +/- 0.2 and 7.6 +/- 0.2) for k(cat)/K(m) as a function of pH was observed. The pH dependence of k(cat) is sigmoidal, described by a single macroscopic pK(a) of 7.7 +/- 0.1, tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 +/- 0.05 mV x C degrees (-1)), and extrapolation to physiologic growth temperature for P. furiosus (100 degrees C) yields a value of - 407 +/- 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic L-proline dehydrogenase. Implications for its mechanism of action are discussed.     

Nonlinear free energy relationship in the general-acid-catalyzed acylation of rat kidney gamma-glutamyl transpeptidase by a series of gamma-glutamyl anilide substrate analogues

The gamma-glutamyl transpeptidase (GGT) purified from rat kidney reacts with a series of eight parasubstituted L-glutamyl gamma-anilides, in the presence of Gly-Gly, catalyzing the formation of gamma-Glu-Gly-Gly (pH 8.0, 37 degrees C). The transpeptidation reaction was followed through the discontinuous colorimetric determination of the concentration of released parasubstituted aniline. Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each anilide substrate. A Hammett plot constructed by the correlation of log(k(cat)) and the sigma(-) parameter for each anilide substrate displays statistically significant upward curvature, consistent with a general-acid-catalyzed acylation mechanism in which the geometry of the transition state changes with the nature of the para substituent. Kinetic isotope effects were measured and are consistent with a reaction involving a proton in flight at the rate-limiting transition state. The pH-rate profiles measured over pH 7.0-9.5 are bell-shaped with kinetic pK(a) values that may be attributed to the active site nucleophile (or its general-base catalytic partner) and the active-site general acid. The variation of the latter pK(a) value as a function of temperature is consistent with an enthalpy of ionization expected for an ammonium ion acting as a general acid. Examination of the variation of k(cat) as a function of temperature gave values for the enthalpy and entropy of activation that are similar to those determined for the general-acid-catalyzed breakdown of the tetrahedral intermediate formed during acylation of chymotrypsin by similar amide substrates.     

Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His(68), His(89), and His(141), identified by affinity labeling and site-directed mutagenesis as critical to the intersubunit catalytic site. The pH-V(max) profile for wild-type ASL is bell-shaped (pK (1) = 6.74 and pK (2) = 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK (2) in the enzyme-substrate complex, we replaced residues at two positions close to His(68) (but not to His(89) or His(141)) in the structure. Compared with the specific activity of 1.75 mumol adenylosuccinate reacting/min/mg of wild-type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has a K (m) for adenylosuccinate similar to that of wild-type ASL, D69N and R310K exhibit modest increases in K (m), and R310Q has an 11-fold increase in K (m). The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH-V(max) profile: pK (2) is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pK of 8.28 observed for wild-type enzyme and attributed to His(68). Similarly, R310Q exhibits a decrease in its pK (2) (7.33), whereas R310K shows little change in pK (2) (8.24). These results suggest that Asp(69) interacts with His(68), that Arg(310) interacts with and orients the beta-carboxylate of Asp(69), and that His(68) must be protonated for ASL to be active.     

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.     

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.