Proton equilibria in the manganese cluster of photosystem II control the intensities of the S(0) and S(2) state g approximately 2 electron paramagnetic resonance signals

We have studied the pH effect on the S(0) and S(2) multiline electron paramagnetic resonance (EPR) signals from the water-oxidizing complex of photosystem II. Around pH 6, the maximum signal intensities were detected. On both the acidic and alkaline sides of pH 6, the intensities of the EPR signals decreased. Two pKs were determined for the S(0) multiline signal; pK(1) = 4.2 +/- 0.2 and pK(2) = 8.0 +/- 0.1, and for the S(2) multiline signal the pKs were pK(1) = 4.5 +/- 0.1 and pK(2) = 7.6 +/- 0.1. The intensity of the S(0)-state EPR signal was partly restored when the pH was changed from acidic or alkaline pH back to pH approximately 6. In the S(2) state we observed partial recovery of the multiline signal when going from alkaline pH back to pH approximately 6, whereas no significant recovery of the S(2) multiline signal was observed when the pH was changed from acidic pH back to pH approximately 6. Several possible explanations for the intensity changes as a function of pH are discussed. Some are ruled out, such as disintegration of the Mn cluster or decay of the S states and formal Cl(-) and Ca(2+) depletion. The altered EPR signal intensities probably reflect the protonation/deprotonation of ligands to the Mn cluster or the oxo bridges between the Mn ions. Also, the possibility of decreased multiline signal intensities at alkaline pH as an effect of changed redox potential of Y(Z) is put forward.     

Activation of the binuclear metal center through formation of phosphotriesterase-inhibitor complexes

Phosphotriesterase (PTE) from Pseudomonas diminuta is a binuclear metalloenzyme that catalyzes the hydrolysis of organophosphate nerve agents at rates approaching the diffusion-controlled limit. The proposed catalytic mechanism postulates the interaction of the substrate with the metal center and subsequent nucleophilic attack by the bridging hydroxide. X-band EPR spectroscopy was utilized to monitor the active site of Mn/Mn-substituted PTE upon addition of two inhibitors, diisopropyl methyl phosphonate and triethyl phosphate, and the product of hydrolysis, diethyl phosphate. The effects of inhibitor and product binding on the magnetic properties of the metal center and the hydroxyl bridge were evaluated by measuring changes in the features of the EPR spectra. The EPR spectra support the proposal that the binding of substrate analogues to the binuclear metal center diminishes the population of hydroxide-bridged species. These results, in conjunction with previously published kinetic and crystallographic data, suggest that substrate binding via the phosphoryl oxygen at the beta-metal weakens the coordination of the hydroxide bridge to the beta-metal. The weakened coordination to the beta-metal ion increases the nucleophilic character of the hydroxide and is coupled to the increase in the electrophilic character of the substrate.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

Implications of ligand binding studies for the catalytic mechanism of cytochrome c oxidase

The reaction of oxidized bovine heart cytochrome c oxidase (CcO) with one equivalent of hydrogen peroxide results in the formation of two spectrally distinct species. The yield of these two forms is controlled by the ionization of a group with a pK(a) of 6.6. At basic pH, where this group is deprotonated, an intermediate called P dominates (P, because it was initially believed to be a peroxy compound). At acidic pH where the group is protonated, a different species, called F (ferryl intermediate) is obtained. We previously proposed that the only difference between these two species is the presence of one proton in the catalytic center of F that is absent in P. It is now suggested that the catalytic center of this F form has the same redox and protonation state as a second ferryl intermediate produced at basic pH by two equivalents of hydrogen peroxide; the role of the second equivalent of H(2)O(2) is that of a proton donor in the conversion of P to F. Two chloride-binding sites have been detected in oxidized CcO. One site is located at the binuclear center; the second site was identified from the sensitivity of g=3 signal of cytochrome a to chloride in the EPR spectra of oxidized CcO. Turnover of CcO releases chloride from the catalytic center into the medium probably by one of the hydrophobic channels, proposed for oxygen access, with an orientation parallel to the membrane plane. Chloride in the binuclear center is most likely not involved in CcO catalysis. The influence of the second chloride site upon several reactions of CcO has been assessed. No correlation was found between chloride binding to the second site and the reactions that were examined.     

Mechanism for the hydrolysis of organophosphates by the bacterial phosphotriesterase

Phosphotriesterase (PTE) from Pseudomonas diminuta is a zinc metalloenzyme that hydrolyzes a variety of organophosphorus compounds. The kinetic parameters of Zn/Zn PTE, Cd/Cd PTE, and a mixed-metal Zn/Cd hybrid PTE were obtained with a variety of substrates to determine the role of each metal ion in binding and catalysis. pH-rate profiles for the hydrolysis of diethyl p-nitrophenyl phosphate (I) and diethyl p-chlorophenyl phosphate (II) demonstrated that the ionization of a single group in the pH range of 5-10 was critical for substrate turnover. The pK(a) values determined from the kinetic assays were dependent on the identity of the metal ion that occupied the alpha site within the binuclear metal center. These results suggest that the hydrolytic nucleophile is activated as a hydroxide via the ionization of a water molecule attached to the alpha-metal ion. The kinetic constants for the hydrolysis of II and diethyl p-chlorophenyl thiophosphate (IV) were determined for the metal substituted forms of PTE. The kinetic constants for IV were greater than those for II. The inverse thio effect is consistent with the polarization of the phosphoryl oxygen/sulfur bond via a direct ligation to the metal center. The rate enhancement is greater when Cd(2+) occupies the beta-metal-ion position. A series of alanine and asparagine mutations were used to characterize the catalytic roles of Asp233, His254, and Asp301. Mutations to either Asp233 or His254 resulted in an enhanced rate of hydrolysis for the sluggish substrate, diethyl p-chlorophenyl phosphate, and a decrease in the kinetic constants for paraoxon (I). These results are consistent with the existence of a proton relay from Asp301 to His254 to Asp233 that is used to ferry protons away from the active site with substrates that do not require activation of the leaving group phenol. A mechanism for the hydrolysis of organophosphates by the bacterial PTE has been proposed.     

Kinetic and chemical mechanisms of the fabG-encoded Streptococcus pneumoniae beta-ketoacyl-ACP reductase

Beta-ketoacyl-acyl carrier protein reductase (KACPR) catalyzes the NADPH-dependent reduction of beta-ketoacyl-acyl carrier protein (AcAc-ACP) to generate (3S)-beta-hydroxyacyl-ACP during the chain-elongation reaction of bacterial fatty acid biosynthesis. We report the evaluation of the kinetic and chemical mechanisms of KACPR using acetoacetyl-CoA (AcAc-CoA) as a substrate. Initial velocity, product inhibition, and deuterium kinetic isotope effect studies were consistent with a random bi-bi rapid-equilibrium kinetic mechanism of KACPR with formation of an enzyme-NADP(+)-AcAc-CoA dead-end complex. Plots of log V/K(NADPH) and log V/K(AcAc)(-)(CoA) indicated the presence of a single basic group (pK = 5.0-5.8) and a single acidic group (pK = 8.0-8.8) involved in catalysis, while the plot of log V vs pH indicated that at high pH an unprotonated form of the ternary enzyme complex was able to undergo catalysis. Significant and identical primary deuterium kinetic isotope effects were observed for V (2.6 +/- 0.4), V/K(NADPH) (2.6 +/- 0.1), and V/K(AcAc)(-)(CoA) (2.6 +/- 0.1) at pH 7.6, but all three values attenuated to values of near unity (1.1 +/- 0.03 or 0.91 +/- 0.02) at pH 10. Similarly, the large alpha-secondary deuterium kinetic isotope effect of 1.15 +/- 0.02 observed for [4R-(2)H]NADPH on V/K(AcAc)(-)(CoA) at pH 7.6 was reduced to a value of unity (1.00 +/- 0.04) at high pH. The complete analysis of the pH profiles and the solvent, primary, secondary, and multiple deuterium isotope effects were most consistent with a chemical mechanism of KACPR that is stepwise, wherein the hydride-transfer step is followed by protonation of the enolate intermediate. Estimations of the intrinsic primary and secondary deuterium isotope effects ((D)k = 2.7, (alpha)(-D)k = 1.16) and the correspondingly negligible commitment factors suggest a nearly full expression of the intrinsic isotope effects on (D)V/K and (alpha)(-D)V/K, and are consistent with a late transition state for the hydride transfer step. Conversely, the estimated intrinsic solvent effect ((D)2(O)k) of 5.3 was poorly expressed in the experimentally derived parameters (D)2(O)V/K and (D)2(O)V (both = 1.2 +/- 0.1), in agreement with the estimation that the catalytic commitment factor for proton transfer to the enolate intermediate is large. Such detailed knowledge of the chemical mechanism of KAPCR may now help guide the rational design of, or inform screening assay-design strategies for, potent inhibitors of this and related enzymes of the short chain dehydrogenase enzyme class.     

The variation of catalytic efficiency of Bacillus cereus metallo-beta-lactamase with different active site metal ions

The kinetics and mechanism of hydrolysis of the native zinc and metal substituted Bacillus cereus (BcII) metallo-beta-lactamase have been investigated. The pH and metal ion dependence of k(cat) and k(cat)/K(m), determined under steady-state conditions, for the cobalt substituted BcII catalyzed hydrolysis of cefoxitin, cephaloridine, and cephalexin indicate that an enzyme residue of apparent pK(a) 6.3 +/- 0.1 is required in its deprotonated form for metal ion binding and catalysis. The k(cat)/K(m) for cefoxitin and cephalexin with cadmium substituted BcII is dependent on two ionizing groups on the enzyme: one of pK(a1) = 8.7 +/- 0.1 required in its deprotonated form and the other of pK(a2) = 9.3 +/- 0.1 required in its protonated form for activity. The pH dependence of the competitive inhibition constant, K(i), for CdBcII with l-captopril indicates that pK(a1) = 8.7 +/- 0.1 corresponds to the cadmium-bound water. For the manganese substituted BcII, the pH dependence of k(cat)/K(m) for benzylpenicillin, cephalexin, and cefoxitin similarly indicated the importance of two catalytic groups: one of pK(a1) = 8.5 +/- 0.1 which needs to be deprotonated and the other of pK(a2) = 9.4 +/- 0.1 which needs to be protonated for catalysis; the pK(a1) was assigned to the manganese-bound water. The rate was metal ion concentration dependent at the highest manganese concentrations used (10(-)(3) M). The metal substituted species have similar or higher catalytic activities compared with the zinc enzyme, albeit at pHs above 7. Interestingly, with cefoxitin, a very poor substrate for ZnBcII, both k(cat) and k(cat)/K(m) increase with increasing pK(a) of the metal-bound water, in the order Zn < Co < Mn < Cd. A higher pK(a) for the metal-bound water for cadmium and manganese BCII leads to more reactive enzymes than the native zinc BcII, suggesting that the role of the metal ion is predominantly to provide the nucleophilic hydroxide, rather than to act as a Lewis acid to polarize the carbonyl group and stabilize the oxyanion tetrahedral intermediate.     

Catalytic mechanism of hamster arylamine N-acetyltransferase 2

Arylamine N-acetyltransferases (NATs) catalyze an acetyl group transfer from AcCoA to primary arylamines, hydrazines, and hydrazides and play a very important role in the metabolism and bioactivation of drugs, carcinogens, and other xenobiotics. The reaction follows a ping-pong bi-bi mechanism. Structure analysis of bacterial NATs revealed a Cys-His-Asp catalytic triad that is strictly conserved in all known NATs. Previously, we have demonstrated by kinetic and isotope effect studies that acetylation of the hamster NAT2 is dependent on a thiolate-imidazolium ion pair (Cys-S(-)-His-ImH(+)) and not a general acid-base catalysis. In addition, we established that, after formation of the acetylated enzyme intermediate, the active-site imidazole, His-107, is likely deprotonated at physiological pH. In this paper, we report steady-state kinetic studies of NAT2 with two acetyl donors, acetyl coenzyme A (AcCoA) and p-nitrophenyl acetate (PNPA), and four arylamine substrates. The pH dependence of k(cat)/K(AcCoA) exhibited two inflection points at 5.32 +/- 0.13 and 8.48 +/- 0.24, respectively. The pK(a) at 5.32 is virtually identical with the previously reported pK(a) of 5.2 for enzyme acetylation, reaffirming that the first half of the reaction is catalyzed by a thiolate-imidazolium ion pair in the active site. The inflection point at 8.48 indicates that a pH-sensitive group on NAT2 is involved in AcCoA binding. A Br?nsted plot constructed by the correlation of log k(4) and log k(H)2(O) with the pK(a) for each arylamine substrate and water displays a linear free-energy relationship in the pK(a) range from -1.7 (H(2)O) to 4.67 (PABA), with a slope of beta(nuc) = 0.80 +/- 0.1. However, a further increase of the pK(a) from 4.67 (PABA) to 5.32 (anisidine) resulted in a 2.5-fold decrease in the k(4) value. Analysis of the pH-k(cat)/K(PABA) profile revealed a pK(a) of 5.52 +/- 0.14 and a solvent kinetic isotope effect (SKIE) of 2.01 +/- 0.04 on k(cat)/K(PABA). Normal solvent isotope effects of 4.8 +/- 0.1, 3.1 +/- 0.1, and 3.2 +/- 0.1 on the k(cat)/K(b) for anisidine, pABglu, and PNA, respectively, were also determined. These observations are consistent with a deacetylation mechanism dominated by nucleophilic attack of the thiol ester for arylamines with pK(a) values or=5.5. The general base is likely His-107 because the His-107 to Gln and Asn mutants were found to be devoid of catalytic activity. In contrast, an increase in pH-dependent hydrolysis of the acetylated enzyme was not observed over a pH range of 5.2-7.5. On the basis of these observations, a catalytic mechanism for the acetylation of arylamines by NAT2 is proposed.     

pH variation of the kinetic parameters and the catalytic mechanism of malic enzyme.

The pH variation of the kinetic parameters for the oxidative decarboxylation of L-malate and decarboxylation of oxalacetate catalyzed by malic enzyme has been used to gain information on the catalytic mechanism of this enzyme. With Mn2+ as the activator, an active-site residue with a pK of 5.4 must be protonated for oxalacetate decarboxylation and ionized for the oxidative decarboxylation of L-malate. With Mg2+ as the metal, this pK is 6, and, at high pH, V/K for L-malate decreases when groups with pKs of 7.8 and 9 are deprotonated. The group at 7.8 is a neutral acid (thought to be water coordinated to Mg2+), while the group at 9 is a cationic acid such as lysine. The V profile for reaction of malate shows these pKs displaced outward by 1.4 pH units, since the rate-limiting step is normally TPNH release, and the chemical reaction, which is pH sensitive, is 25 times faster. TPN binding is decreased by ionization of a group with pK 9.3 or protonation of a group with pK 5.3. The pH variation of the Km for Mg shows that protonation of a group with pK 8.7 (possibly SH) decreases metal binding in the presence of malate by a factor of 1400, and in the absence of malate by a factor of 20. A catalytic mechanism is proposed in which hydride transfer is accompanied by transfer of a proton to the group with pK 5.4-6, and enolpyruvate is protonated by water coordinated to the Mg2+ (pK 7.8) after decarboxylation and release of CO2.     

Kinetic studies of guinea pig liver transglutaminase reveal a general-base-catalyzed deacylation mechanism.

Guinea pig liver transglutaminase (TGase) reacts with 0.1 mM N-Cbz-L-Glu(gamma-p-nitrophenyl ester)Gly (5, prepared herein, K(M) = 0.02 mM) to undergo rapid acylation that can be followed spectrophotometrically at 400 nm (pH 7.0, 25 degrees C). Deacylation of the transiently formed thiolester acyl enzyme intermediate via catalytic aminolysis was studied in the presence of six primary amines of widely varying basicity (pK(NH+) = 5.6-10.5). Steady-state kinetic studies were performed to measure k(cat) and K(M) values for each amine substrate. A Br?nsted plot constructed through the correlation of log(k(cat)/K(M)) and pK(NH+) for each amine substrate displays a linear free-energy relationship with a slope beta(nuc) = -0.37 +/- 0.08. The shallow negative slope is consistent with a general-base-catalyzed deacylation mechanism in which a proton is removed from the amine substrate during its rate-limiting nucleophilic attack on the thiolester carbonyl. Kinetic isotope effects were measured for four acceptor substrates (water, kie = 1.1 +/- 0.1; aminoacetonitrile, kie = 5.9 +/- 1.2; glycine methyl ester, kie = 3.4 +/- 0.7; N-Ac-L-lysine methyl ester, kie = 1.1 +/- 0.1) and are consistent with a proton in flight at the rate-limiting transition state. The active site general-base implicated by these kinetic results is believed to be His-334, of the highly conserved TGase Cys-His-Asp catalytic triad.     

The pH dependence of the reductive carboxylation of pyruvate by malic enzyme

The maximum velocity of the malic enzyme (L-malate: NADP+ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40) reductive carboxylation of pyruvate and V/KCO2 are pH-independent from pH 5.5 to pH 8.5. V/K for pyruvate exhibits pK values values of 6.50 +/- 0.25 and 7.25 +/- 0.25. These data suggest that the binding of pyruvate locks the protonation state of enzyme. In addition, the pK values are within experimental error identical for the pH dependence of V/Kmalate and V/Kpyruvate. Thus, the catalytic groups appear to have reverse protonation states in the two reaction directions. The ratio of (V/Kmalate)/(V/Kpyruvate) is 100, suggesting that the protonation state of enzyme is optimum in the malate oxidative decarboxylation direction. Thus, the group with a pK of about 6 is unprotonated and the group with a pK of 7.5 is protonated for malate decarboxylation, and the opposite is true for pyruvate reductive carboxylation.     

Enzyme deactivation due to metal-ion dissociation during turnover of the cobalt-beta-lactamase catalyzed hydrolysis of beta-lactams

Metallo-beta-lactamases are native zinc enzymes that catalyze the hydrolysis of beta-lactam antibiotics but are also able to function with cobalt (II) and require one or two metal ions for catalytic activity. The kinetics of the hydrolysis of benzylpenicillin catalyzed by cobalt substituted beta-lactamase from Bacillus cereus (BcII) are biphasic. The dependence of enzyme activity on pH and metal-ion concentration indicates that only the di-cobalt enzyme is catalytically active. A mono-cobalt enzyme species is formed during the catalytic cycle, which is virtually inactive and requires the association of another cobalt ion for turnover. Two intermediates with different metal to enzyme stoichiometries are formed on a branched reaction pathway. The di-cobalt enzyme intermediate is responsible for the direct catalytic route, which is pH-independent between 5.5 and 9.5 but is also able to slowly lose one bound cobalt ion via the branching route to give the mono-cobalt inactive enzyme intermediate. This inactivation pathway of metal-ion dissociation occurs by both an acid catalyzed and a pH-independent reaction, which is dependent on the presence of an enzyme residue of pK(a) = 8.9 +/- 0.1 in its protonated form and shows a large kinetic solvent isotope effect (H(2)O/D(2)O) of 5.2 +/- 0.5, indicative of a rate-limiting proton transfer. The pseudo first-order rate constant to regenerate the di-cobalt beta-lactamase from the mono-cobalt enzyme intermediate has a first-order dependence on cobalt-ion concentration in the pH range 5.5-9.5. The second-order rate constant for metal-ion association is dependent on two groups of pK(a) 6.32 +/- 0.1 and 7.47 +/- 0.1 being in their deprotonated basic forms and one group of pK(a) 9.48 +/- 0.1 being in its protonated form.     

Self-assembly of the binuclear metal center of phosphotriesterase

The active site of the bacterial phosphotriesterase (PTE) from Pseudomonas diminuta contains two divalent metal ions and a carboxylated lysine residue. The native enzyme contains two Zn(2+) ions, which can be replaced with Co(2+), Cd(2+), Ni(2+), or Mn(2+) without loss of catalytic activity. Carbon dioxide reacts with the side chain of lysine-169 to form a carbamate functional group within the active site, which then serves as a bridging ligand to the two metal ions. The activation of apo-PTE using variable concentrations of divalent metal ions and bicarbonate was measured in order to establish the mechanism by which the active site of PTE is self-assembled. The time courses for the activation of apo-PTE are pseudo-first-order, and the observed rate constants are directly proportional to the concentration of bicarbonate. In contrast, the apparent rate constants for the activation of apo-PTE decrease as the concentrations of the divalent cations are increased and then become constant at higher concentrations of the divalent metal ions. These results are consistent with a largely ordered kinetic mechanism for the assembly of the binuclear metal center where CO(2)/bicarbonate reacts with the apo-PTE prior to the binding of the two metal ions. When apo-PTE is titrated with 0-8 equiv of Co(2+), Cd(2+), or Zn(2+), the concentration of activated enzyme increases linearly until 2 equiv of metal ion is added and then remains constant at elevated levels of the divalent cations. These results are consistent with the synergistic binding of the two metal ions to the active site, and thus the second metal ion binds more tightly to the protein than does the first metal ion. Measurement of the mean dissociation constant indicates that metal binding to the binuclear metal center is strong [(K(alpha)K(beta))(1/2) = 6.0 x 10(-)(11) M and k(off) = 1.5 x 10(-)(3) min(-)(1) for Zn(2+)]. The removal of the carbamate bridge through the mutagenesis of Lys-169 demonstrates that the carbamate bridge is required for both efficient catalysis and overall stability of the metal center.     

Protonation reactions in relation to the coupling mechanism of bovine cytochrome c oxidase

Identification of the locations of protonatable sites in cytochrome c oxidase that are influenced by reactions in the binuclear centre is critical to assessment of proposed coupling mechanisms, and to controversies on where the pumping steps occur. One such protonation site is that which governs interconversion of the isoelectronic 607 nm 'P(M)' and 580 nm 'F' forms of the two-electron-reduced oxygen intermediate. Low pH favours protonation of a site that is close to an electron paramagnetic resonance (EPR)-silent radical species in P(M), and this induces a partial electronic redistribution to form an EPR-detectable tryptophan radical in F. A further protonatable group that must be close to the binuclear centre has been detected in bacterial oxidases by Fourier transform infrared spectroscopy from pH-dependent changes in the haem-bound CO vibration frequency at low temperatures. However, in bovine cytochrome c oxidase under similar conditions of measurement, haem-bound CO remains predominantly in a single 1963 cm(-1) form between pH 6.5 and 8.5, indicating that this group is not present. Lack of pH dependence extends to the protein region of the CO photolysis spectra and suggests that both the reduced and the reduced/CO states do not have titratable groups that affect the binuclear centre strongly in the pH range 6.5-8.5. This includes the conserved glutamic acid residue E242 whose pK appears to be above 8.5 even in the fully oxidised enzyme. The results are discussed in relation to recent ideas on coupling mechanism.     

Mutational,kinetic, and NMR studies of the mechanism of E. coli GDP-mannose mannosyl hydrolase,an unusual Nudix enzyme

GDP-mannose mannosyl hydrolase (GDPMH) is an unusual Nudix family member, which catalyzes the hydrolysis of GDP-alpha-D-mannose to GDP and the beta-sugar by nucleophilic substitution at carbon rather than at phosphorus (Legler, P. M., Massiah, M. A., Bessman, M. J., and Mildvan, A. S. (2000) Biochemistry 39, 8603-8608). Using the structure and mechanism of MutT, the prototypical Nudix enzyme as a guide, we detected six catalytic residues of GDPMH, three of which were unique to GDPMH, by the kinetic and structural effects of site-specific mutations. Glu-70 (corresponding to Glu-57 in MutT) provides a ligand to the essential divalent cation on the basis of the effects of the E70Q mutation which decreased kcat 10(2.2)-fold, increased the dissociation constant of Mn2+ from the ternary E-Mn2+-GDP complex 3-fold, increased the K(m)Mg2+ 20-fold, and decreased the paramagnetic effect of Mn2+ on 1/T1 of water protons, indicating a change in the coordination sphere of Mn2+. In the E70Q mutant, Gln-70 was shown to be very near the active site metal ion by large paramagnetic effects of Mn2+ on its side chain -NH2 group. With wild-type GDPMH, the effect of pH on log(kcat/K(m)GDPmann) at 37 degrees C showed an ascending limb of unit slope, followed by a plateau yielding a pK(a) of 6.4, which increased to 6.7 +/- 0.1 in the pH dependence of log(kcat). The general base catalyst was identified as a neutral His residue by the DeltaH(ionization) = 7.0 +/- 0.7 kcal/mol, by the increase in pK(a) with ionic strength, and by mutation of each of the four histidine residues of GDPMH to Gln. Only the H124Q mutant showed the loss of the ascending limb in the pH versus log(kcat) rate profile, which was replaced by a weak dependence of rate on hydroxide concentration, as well as an overall 10(3.4)-fold decrease in kcat, indicating His-124 to be the general base, unlike MutT, which uses Glu-53 in this role. The H88Q mutant showed a 10(2.3)-fold decrease in kcat, a 4.4-fold increase in K(m)GDPmann, and no change in the pH versus log(kcat) rate profile, indicating an important but unidentified role of His-88 in catalysis. One and two-dimensional NMR studies permitted the sequence specific assignments of the imidazole HdeltaC, H(epsilon)C, N(delta), and N(epsilon) resonances of the four histidines and defined their protonation states. The pK(a) of His-124 (6.94 +/- 0.04) in the presence of saturating Mg2+ was comparable to the kinetically determined pK(a) at the same temperature (6.40 +/- 0.20). The other three histidines were neutral N(epsilon)H tautomers with pK(a) values below 5.5. Arg-52 and Arg-65 were identified as catalytic residues which interact electrostatically with the GDP leaving group by mutating these residues to Gln and Lys. The R52Q mutant decreased kcat 309-fold and increased K(m)GDPmann 40.6-fold, while the R52K mutant decreased kcat by only 12-fold and increased K(m)GDPmann 81-fold. The partial rescue of kcat, but not of K(m)GDPmann in the R52K mutant, suggests that Arg-52 is a bifunctional hydrogen bond donor to the GDP leaving group in the ground state and a monofunctional hydrogen bond donor in the transition state. Opposite behavior was found with the Arg-65 mutants, suggesting this residue to be a monofunctional hydrogen bond donor to the GDP leaving group in the ground state and a bifunctional hydrogen bond donor in the transition state. From these observations, a mechanism for GDPMH is proposed involving general base catalysis and electrostatic stabilization of the leaving group.     

Water-hydroxide exchange reactions at the catalytic site of heme-copper oxidases

Membrane-bound heme-copper oxidases catalyze the reduction of O(2) to water. Part of the free energy associated with this process is used to pump protons across the membrane. The O(2) reduction reaction results in formation of high-pK(a) protonatable groups at the catalytic site. The free energy associated with protonation of these groups is used for proton pumping. One of these protonatable groups is OH(-), coordinated to the heme and Cu(B) at the catalytic site. Here we present results from EPR experiments on the Rhodobacter sphaeroides cytochrome c oxidase, which show that at high pH (9) approximately 50% of oxidized heme a(3) is hydroxide-ligated, while at low pH (6.5), no hydroxide is bound to heme a(3). The kinetics of hydroxide binding to heme a(3) were investigated after dissociation of CO from heme a(3) in the enzyme in which the heme a(3)-Cu(B) center was reduced while the remaining redox sites were oxidized. The dissociation of CO results in a decrease of the midpoint potential of heme a(3), which results in electron transfer (tau approximately equal 3 micros) from heme a(3) to heme a in approximately 100% of the enzyme population. At pH >7.5, the electron transfer is followed by proton release from a H(2)O molecule to the bulk solution (tau approximately equal 2 ms at pH 9). This reaction is also associated with absorbance changes of heme a(3), which on the basis of the results from the EPR experiments are attributed to formation of hydroxide-ligated heme a(3). The OH(-) bound to heme a(3) under equilibrium conditions at high pH is also formed transiently after O(2) reduction at low pH. It is proposed that the free energy associated with electron transfer to the binuclear center and protonation of this OH(-) upon reduction of the recently oxidized enzyme provides the driving force for the pumping of one proton.     

The reaction mechanism of paraoxon hydrolysis by phosphotriesterase from combined QM/MM simulations

Molecular dynamics simulations employing combined quantum mechanical and molecular mechanical (QM/MM) potentials have been carried out to investigate the reaction mechanism of the hydrolysis of paraoxon by phosphotriesterase (PTE). We used a dual-level QM/MM approach that synthesizes accurate results from high-level electronic structure calculations with computational efficiency of semiempirical QM/MM potentials for free energy simulations. In particular, the intrinsic (gas-phase) energies of the active site in the QM region are determined by using density functional theory (B3LYP) and second-order M?ller-Plesset perturbation theory (MP2) and the molecular dynamics free energy simulations are performed by using the mixed AM1:CHARMM potential. The simulation results suggest a revised mechanism for the phosphotriester hydrolysis mechanism by PTE. The reaction free energy profile is mirrored by structural motions of the binuclear metal center in the active site. The two zinc ions occupy a compact conformation with an average zinc-zinc distance of 3.5 +/- 0.1 A in the Michaelis complex, whereas it is elongated to 5.3 +/- 0.3 A at the transition state and product state. The substrate is loosely bound to the more exposed zinc ion (Znbeta2+) at an average distance of 3.8 A +/- 0.3 A. The P=O bond of the substrate paraoxon is activated by adopting a tight coordination to the Znbeta2+, releasing the coordinate to the bridging hydroxide ion and increasing its nucleophilicity. It was also found that a water molecule enters into the binding pocket of the loosely bound binuclear center, originally occupied by the nucleophilic hydroxide ion. We suggest that the proton of this water molecule is taken up by His254 at low pH or released to the solvent at high pH, resulting in a hydroxide ion that pulls the Znbeta2+ ion closer to form the compact configuration and restores the resting state of the enzyme.     

Reverse protonation is the key to general acid-base catalysis in enolase

The pH dependence of enolase catalysis was studied to understand how enolase is able to utilize both general acid and general base catalysis in each direction of the reaction at near-neutral pHs. Wild-type enolase from yeast was assayed in the dehydration reaction (2-phospho-D-glycerate --> phosphoenolpyruvate + H(2)O) at different pHs. E211Q, a site-specific variant of enolase that catalyzes the exchange of the alpha-proton of 2-phospho-D-glycerate but not the complete dehydration, was assayed in a (1)H/(2)H exchange reaction at different pDs. Additionally, crystal structures of E211Q and E168Q were obtained at 2.0 and 1.8 A resolution, respectively. Analysis of the pH profile of k(cat)/K(Mg) for wild-type enolase yielded macroscopic pK(a) estimates of 7.4 +/- 0.3 and 9.0 +/- 0.3, while the results of the pD profile of the exchange reaction of E211Q led to a pK(a) estimate of 9.5 +/- 0.1. These values permit estimates of the four microscopic pK(a)s that describe the four relevant protonation states of the acid/base catalytic groups in the active site. The analysis indicates that the dehydration reaction is catalyzed by a small fraction of enzyme that is reverse-protonated (i.e., Lys345-NH(2), Glu211-COOH), whereas the hydration reaction is catalyzed by a larger fraction of the enzyme that is typically protonated (i.e., Lys345-NH(3)(+), Glu211-COO(-)). These two forms of the enzyme coexist in a constant, pH-independent ratio. The structures of E211Q and E168Q both show virtually identical folds and active-site architectures (as compared to wild-type enolase) and thus provide additional support to the conclusions reported herein. Other enzymes that require both general acid and general base catalysis likely require reverse protonation of catalytic groups in one direction of the reaction.     

The kinetics of the reactions of Parasponia andersonii hemoglobin with oxygen, carbon monoxide, and nitric oxide

Hemoglobin I was isolated from nodules formed on the roots of Parasponia andersonii inoculated with Rhizobium strain CP 283. The rate of oxygen dissociation from Parasponia hemoglobin increases about 12-fold between pH 4 and 7, with apparent pK 6.4, to reach a limiting value of 14.8s-1. The optical spectrum of oxyhemoglobin in the visible region is also dependent on pH with pK near 6.4. The rate constant for oxygen combination with Parasponia hemoglobin increases about 7-8-fold between pH 4 and 7, with apparent pK 5.37, to reach a value of 1.67 X 10(8) M-1 s-1 at pH 7. The optical spectrum of deoxyhemoglobin in the visible region and the rate constant for carbon monoxide combination are also dependent on pH with apparent pK 5.65 and 5.75, respectively. The rate constant for carbon monoxide dissociation is independent of pH. The oxygen affinity of Parasponia hemoglobin, P50 = 0.049 torr at 20 degrees C, calculated from the kinetic constants at pH 7, is very great. At alkaline pH there is a prominent geminate reaction with oxygen and nitric oxide, with both subnanosecond and tens of nanosecond components. These reactions disappear at acid pH, with pK 6.4, and the effective quantum yield is reduced. In general, the reactions of Parasponia hemoglobin with oxygen and carbon monoxide resemble those of soybean leghemoglobin. In each, great oxygen affinity is achieved by unusually rapid oxygen combination together with a moderate rate of oxygen dissociation. We suggest that protonation of a heme-linked group with pK near 6.4 controls many properties of Parasponia oxyhemoglobin, and protonation of a group with pK near 5.5 controls many properties of Parasponia deoxyhemoglobin.     

Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154-->Cys forms of yeast xylitol dehydrogenase

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10-0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19+/-0.03 s(-1) and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019+/-0.003% and 0.74+/-0.03% of wild-type catalytic efficiency (kcat/K(sorbitol)=7800+/-700 M(-1) x s(-1)) and kcat (=161+/-4 s(-1)) for NAD+-dependent oxidation of sorbitol at 25 degrees C respectively. The pH profile of kcat/K(sorbitol) for E154C decreased below an apparent pK of 9.1+/-0.3, reflecting a shift in pK by about +1.7-1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (approximately +0.2 log units), suggesting that the observed pK in the binary enzyme-NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7+/-0.2 (E154C, 1.7+/-0.1) and 1.9+/-0.3 (E154C, 2.4+/-0.2) on kcat/K(sorbitol) respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687+/-12 s(-1) in the pre-steady state, which features a turnover of 0.9+/-0.1 enzyme equivalents as NADH was produced with a rate constant of 409+/-3 s(-1). The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.