UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase functions through a general acid-base catalyst pair mechanism

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of k(cat)/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4+/-0.1 and 9.1+/-0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile.     

Staphylococcus aureus sortase transpeptidase SrtA: insight into the kinetic mechanism and evidence for a reverse protonation catalytic mechanism

The Staphylococcus aureus transpeptidase SrtA catalyzes the covalent attachment of LPXTG-containing virulence and colonization-associated proteins to cell-wall peptidoglycan in Gram-positive bacteria. Recent structural characterizations of staphylococcal SrtA, and related transpeptidases SrtB from S. aureus and Bacillus anthracis, provide many details regarding the active site environment, yet raise questions with regard to the nature of catalysis and active site cysteine thiol activation. Here we re-evaluate the kinetic mechanism of SrtA and shed light on aspects of its catalytic mechanism. Using steady-state, pre-steady-state, bisubstrate kinetic studies, and high-resolution electrospray mass spectrometry, revised steady-state kinetic parameters and a ping-pong hydrolytic shunt kinetic mechanism were determined for recombinant SrtA. The pH dependencies of kinetic parameters k(cat)/K(m) and k(cat) for the substrate Abz-LPETG-Dap(Dnp)-NH(2) were bell-shaped with pK(a) values of 6.3 +/- 0.2 and 9.4 +/- 0.2 for k(cat) and 6.2 +/- 0.2 and 9.4 +/- 0.2 for k(cat)/K(m). Solvent isotope effect (SIE) measurements revealed inverse behavior, with a (D)2(O)k(cat) of 0.89 +/- 0.01 and a (D)2(O)(k(cat)/K(m)) of 0.57 +/- 0.03 reflecting an equilibrium SIE. In addition, SIE measurements strongly implicated Cys184 participation in the isotope-sensitive rate-determining chemical step when considered in conjunction with an inverse linear proton inventory for k(cat). Last, the pH dependence of SrtA inactivation by iodoacetamide revealed a single ionization for inactivation. These studies collectively provide compelling evidence for a reverse protonation mechanism where a small fraction (ca. 0.06%) of SrtA is competent for catalysis at physiological pH, yet is highly active with an estimated k(cat)/K(m) of >10(5) M(-)(1) s(-)(1).     

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.     

Acid-base catalysis by UDP-galactose 4-epimerase: correlations of kinetically measured acid dissociation constants with thermodynamic values for tyrosine 149

The steady-state kinetic parameters for epimerization of UDP-galactose by UDP-galactose 4-epimerase from Escherichia coli (GalE), Y149F-GalE, and S124A-GalE have been measured as a function of pH. The deuterium kinetic isotope effects for epimerization of UDP-galactose-C-d(7) by these enzymes have also been measured. The results show that the activity of wild-type GalE is pH-independent in the pH range of 5.5-9.3, and there is no significant deuterium kinetic isotope effect in the reaction of UDP-galactose-C-d(7). It is concluded that the rate-limiting step for epimerization by wild-type GalE is not hydride transfer and must be either a diffusional process or a conformational change. Epimerization of UDP-galactose-C-d(7) by Y149F-GalE proceeds with a pH-dependent deuterium kinetic isotope effect on k(cat) of 2.2 +/- 0.4 at pH 6.2 and 1.1 +/- 0.5 at pH 8.3. Moreover, the plot of log k(cat)/K(m) breaks downward on the acid side with a fitted value of 7.1 for the pK(a). It is concluded that the break in the pH-rate profile arises from a change in the rate-limiting step from hydride transfer at low pH to a conformational change at high pH. Epimerization of UDP-galactose-C-d(7) by S124A-GalE proceeds with a pH-independent deuterium kinetic isotope effect on k(cat) of 2.0 +/- 0.2 between pH 6 and 9. Both plots of log k(cat) and log k(cat)/K(m) display pH dependence. The plot of log k(cat) versus pH breaks downward with a pK(a) of 6.35 +/- 0.10. The plot of log k(cat)/K(m) versus pH is bell-shaped, with fitted pK(a) values of 6.76 +/- 0.09 and 9.32 +/- 0.21. It is concluded that hydride transfer is rate-limiting, and the pK(a) of 6.7 for free S124A-GalE is assigned to Tyr 149, which displays the same value of pK(a) when measured spectrophotometrically in this variant. Acid-base catalysis by Y149F-GalE is attributed to Ser 124, which is postulated to rescue catalysis of proton transfer in the absence of Tyr 149. The kinetic pK(a) of 7.1 for free Y149F-GalE is lower than that expected for Ser 124, as proven by the pH-dependent kinetic isotope effect. Epimerization by the doubly mutated Y149F/S124A-GalE proceeds at a k(cat) that is lower by a factor of 10(7) than that of wild-type GalE. This low rate is attributed to the synergistic actions of Tyr 149 and Ser 124 in wild-type GalE and to the absence of any internal catalysis of hydride transfer in the doubly mutated enzyme.     

Inhibition and pH dependence of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the NAD-dependent oxidation of phosphite to phosphate, a reaction that is 15 kcal/mol exergonic. The enzyme belongs to the family of D-hydroxy acid dehydrogenases. Five other family members that were analyzed do not catalyze the oxidation of phosphite, ruling out the possibility that this is a ubiquitous activity of these proteins. PTDH does not accept any alternative substrates such as thiophosphite, hydrated aldehydes, and methylphosphinate, and potential small nucleophiles such as hydroxylamine, fluoride, methanol, and trifluoromethanol do not compete with water in the displacement of the hydride from phosphite. The pH dependence of k(cat)/K(m,phosphite) is bell-shaped with a pK(a) of 6.8 for the acidic limb and a pK(a) of 7.8 for the basic limb. The pK(a) of 6.8 is assigned to the second deprotonation of phosphite. However, whether the dianionic form of phosphite is the true substrate is not clear since a reverse protonation mechanism is also consistent with the available data. Unlike k(cat)/K(m,phosphite), k(cat) and k(cat)/K(m,NAD) are pH-independent. Sulfite is a strong inhibitor of PTDH that is competitive with respect to phosphite and uncompetitive with respect to NAD(+). Incubation of the enzyme with NAD(+) and low concentrations of sulfite results in a covalent adduct between NAD(+) and sulfite in the active site of the enzyme that binds very tightly. Fluorescent titration studies provided the apparent dissociation constants for NAD(+), NADH, sulfite, and the sulfite-NAD(+) adduct. Substrate isotope effect studies with deuterium-labeled phosphite resulted in small normal isotope effects (1.4-2.1) on both k(cat) and k(cat)/K(m,phosphite) at pH 7.25 and 8.0. Solvent isotope effects (SIEs) on k(cat) are similar in size; however, the SIE of k(cat)/K(m,phosphite) at pH 7.25 is significantly larger (4.4), whereas at pH 8.0, it is the inverse (0.6). The pH-rate profile of k(cat)/K(m,phosphite), which predicts that the observed SIEs will have a significant thermodynamic origin, can account for these effects.     

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.     

Refined solution structure of the LpxC-TU-514 complex and pKa analysis of an active site histidine: insights into the mechanism and inhibitor design

Lipopolysaccharide, the major constituent of the outer monolayer of the outer membrane of Gram-negative bacteria, is anchored into the membrane through the hydrophobic moiety lipid A, a hexaacylated disaccharide. The zinc-dependent metalloamidase UDP-3-O-acyl-N-acetylglucosamine deacetylase (LpxC) catalyzes the second and committed step in the biosynthesis of lipid A. LpxC shows no homology to mammalian metalloamidases and is essential for cell viability, making it an important target for the development of novel antibacterial compounds. Recent NMR and X-ray studies of the LpxC from Aquifex aeolicus have provided the first structural information about this family of proteins. Insight into the catalytic mechanism and the design of effective inhibitors could be facilitated by more detailed structural and biochemical studies that define substrate-protein interactions and the roles of specific residues in the active site. Here, we report the synthesis of the (13)C-labeled substrate-analogue inhibitor TU-514, and the subsequent refinement of the solution structure of the A. aeolicus LpxC-TU-514 complex using residual dipolar couplings. We also reevaluate the catalytic role of an active site histidine, H253, on the basis of both its pK(a) as determined by NMR titration and pH-dependent kinetic analyses. These results provide a structural basis for the design of more potent LpxC inhibitors than those that are currently available.     

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.     

Discovery and characterization of human antibody inhibitors of pregnancy-associated plasma protein-A

Pregnancy-associated plasma protein-A (PAPP-A) is a metalloprotease that cleaves insulin-like growth factor-binding proteins (IGFBPs) to release bioactive levels of free insulin-like growth factor. Specific and potent inhibitors of PAPP-A may further elucidate the biological functions of this protease and could prove to be of therapeutic value. Phage display was used to discover fully human antibody inhibitors of PAPP-A activity towards IGFBP4 cleavage. Estimates of the inhibition constants for these antibodies were subsequently determined using a novel continuous assay of PAPP-A protease activity that uses an internally quenched synthetic peptide substrate (DX-1655). DX-1655 was hydrolyzed by PAPP-A with a K(m) of 33 muM and a k(cat) of 0.3 s(-1) (k(cat)/K(m)=9.1x10(3) M(-1) s(-1)). PAPP-A activity towards DX-1655 displays a bell-shaped pH profile, with pK(a) values of 8.2 and 10.8 and a maximum rate at approximately pH 9.5. Using this continuous assay, we measured apparent K(i) values of 1.7+/-0.2 and 7.4+/-1.5 nM for the F2 and D9 antibodies, respectively.     

Effects of mutations of the active site arginine residues in 4-oxalocrotonate tautomerase on the pKa values of active site residues and on the pH dependence of catalysis.

The unusually low pK(a) value of the general base catalyst Pro-1 (pK(a) = 6.4) in 4-oxalocrotonate tautomerase (4-OT) has been ascribed to both a low dielectric constant at the active site and the proximity of the cationic residues Arg-11 and Arg-39 [Stivers, J. T., Abeygunawardana, C., Mildvan, A. S., Hajipour, G., and Whitman, C. P. (1996) Biochemistry 35, 814-823]. In addition, the pH-rate profiles in that study showed an unidentified protonated group essential for catalysis with a pK(a) of 9.0. To address these issues, the pK(a) values of the active site Pro-1 and lower limit pK(a) values of arginine residues were determined by direct (15)N NMR pH titrations. The pK(a) values of Pro-1 and of the essential acid group were determined independently from pH-rate profiles of the kinetic parameters of 4-OT in arginine mutants of 4-OT and compared with those of wild type. The chemical shifts of all of the Arg Nepsilon resonances in wild-type 4-OT and in the R11A and R39Q mutants were found to be independent of pH over the range 4.9-9.7, indicating that no arginine is responsible for the kinetically determined pK(a) of 9.0 for an acidic group in free 4-OT. With the R11A mutant, where k(cat)/K(m) was reduced by a factor of 10(2.9), the pK(a) of Pro-1 was not significantly altered from that of the wild-type enzyme (pK(a) = 6.4 +/- 0.2) as revealed by both direct (15)N NMR titration (pK(a) = 6.3 +/- 0.1) and the pH dependence of k(cat)/K(m) (pK(a) = 6.4 +/- 0.2). The pH-rate profiles of both k(cat)/K(m) and k(cat) for the reaction of the R11A mutant with the dicarboxylate substrate, 2-hydroxymuconate, showed humps, i.e., sharply defined maxima followed by nonzero plateaus. The humps disappeared in the reaction with the monocarboxylate substrate, 2-hydroxy-2,4-pentadienoate, indicating that, unlike the wild-type enzyme which reacts only with the dianionic form of the dicarboxylic substrate, the R11A mutant reacts with both the 6-COOH and 6-COO(-) forms, with the 6-COOH form being 12-fold more active. This reversal in the preferred ionization state of the 6-carboxyl group of the substrate that occurs upon mutation of Arg-11 to Ala provides strong evidence that Arg-11 interacts with the 6-carboxylate of the substrate. In the R39Q mutant, where k(cat)/K(m) was reduced by a factor of 10(3), the kinetically determined pK(a) value for Pro-1 was 4.6 +/- 0.2, while the ionization of Pro-1 showed negative cooperativity with an apparent pK(a) of 7.1 +/- 0.1 determined by 1D (15)N NMR. From the Hill coefficient of 0.54, it can be shown that the apparent pK(a) value of 7.1 could result most simply from the averaging of two limiting pK(a) values of 4.6 and 8.2. Mutation of Arg-39, by altering the structure of the beta-hairpin which covers the active site, could result in an increase in the solvent exposure of Pro-1, raising its upper limit pK(a) value to 8.2. In the R39A mutant, the kinetically determined pK(a) of Pro-1 was also low, 5.0 +/- 0.2, indicating that in both the R39Q and R39A mutants, only the sites with low pK(a) values were kinetically operative. With the fully active R61A mutant, the kinetically determined pK(a) of Pro-1 (pK(a) = 6.5 +/- 0.2) agreed with that of wild-type 4-OT. It is concluded that the unusually low pK(a) of Pro-1 shows little contribution from electrostatic effects of the nearby cationic Arg-11, Arg-39, and Arg-61 residues but results primarily from a site of low local dielectric constant.     

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.     

Antibacterial agents that target lipid A biosynthesis in gram-negative bacteria. Inhibition of diverse UDP-3-O-(r-3-hydroxymyristoyl)-n-acetylglucosamine deacetylases by substrate analogs containing zinc binding motifs

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) catalyzes the second step in the biosynthesis of lipid A, a unique amphiphilic molecule found in the outer membranes of virtually all Gram-negative bacteria. Since lipid A biosynthesis is required for bacterial growth, inhibitors of LpxC have potential utility as antibiotics. The enzymes of lipid A biosynthesis, including LpxC, are encoded by single copy genes in all sequenced Gram-negative genomes. We have now cloned, overexpressed, and purified LpxC from the hyperthermophile Aquifex aeolicus. This heat-stable LpxC variant (the most divergent of all known LpxCs) displays 32% identity and 51% similarity over 277 amino acid residues out of the 305 in Escherichia coli LpxC. Although A. aeolicus LpxC deacetylates the substrate UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine at a rate comparable with E. coli LpxC, a phenyloxazoline-based hydroxamate that inhibits E. coli LpxC with K(i) of approximately 50 nM (Onishi, H. R., Pelak, B. A., Gerckens, L. S., Silver, L. L., Kahan, F. M., Chen, M. H., Patchett, A. A., Galloway, S. M., Hyland, S. A., Anderson, M. S., and Raetz, C. R. H. (1996) Science 274, 980-982) does not inhibit A. aeolicus LpxC. To determine whether or not broad-spectrum deacetylase inhibitors can be found, we have designed a new class of hydroxamate-containing inhibitors of LpxC, starting with the structure of the physiological substrate. Several of these compounds inhibit both E. coli and A. aeolicus LpxC at similar concentrations. We have also identified a phosphinate-containing substrate analog that inhibits both E. coli and A. aeolicus LpxC, suggesting that the LpxC reaction proceeds by a mechanism similar to that described for other zinc metalloamidases, like carboxypeptidase A and thermolysin. The differences between the phenyloxazoline and the substrate-based LpxC inhibitors might be exploited for developing novel antibiotics targeted either against some or all Gram-negative strains. We suggest that LpxC inhibitors with antibacterial activity be termed "deacetylins."     

²H kinetic isotope effects and pH dependence of catalysis as mechanistic probes of rat monoamine oxidase A: comparisons with the human enzyme

Monoamine oxidase A (MAO A) is a mitochondrial outer membrane-bound flavoenzyme important in the regulation of serotonin and dopamine levels. Because the rat is extensively used as an animal model in drug studies, it is important to understand how rat MAO A behaves in comparison with the more extensively studied human enzyme. For many reversible inhibitors, rat MAO A exhibits K(i) values similar to those of human MAO A. The pH profile of k(cat) for rat MAO A shows a pK(a) of 8.2 ± 0.1 for the benzylamine ES complex and pK(a) values of 7.5 ± 0.1 and 7.6 ± 0.1 for the ES complexes with p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine, respectively. In contrast to the human enzyme, the rat enzyme exhibits a single pK(a) value (8.3 ± 0.1) with k(cat)/K(m) for benzylamine versus pH and pK(a) values of 7.8 ± 0.1 and 8.1 ± 0.2 for the ascending limbs, respectively, of k(cat)/K(m) versus pH profiles for p-CF(3)-(1)H- and p-CF(3)-(2)H-benzylamine and 9.3 ± 0.1 and 9.1 ± 0.2 for the descending limbs, respectively. The oxidation of para-substituted benzylamine substrate analogues by rat MAO A has large deuterium kinetic isotope effects on k(cat) and on k(cat)/K(m). These effects are pH-independent and range from 7 to 14, demonstrating a rate-limiting α-C-H bond cleavage step in catalysis. Quantitative structure-activity correlations of log k(cat) with the electronic substituent parameter (σ) at pH 7.5 and 9.0 show a dominant contribution with positive ρ values (1.2-1.3) and a pH-independent negative contribution from the steric term. Quantitative structure-activity relationship analysis of the binding affinities of the para-substituted benzylamine analogues for rat MAO A shows an increased van der Waals volume (V(w)) increases the affinity of the deprotonated amine for the enzyme. These results demonstrate that rat MAO A exhibits functional properties similar but not identical with those of the human enzyme and provide additional support for C-H bond cleavage via a polar nucleophilic mechanism.     

Solvent isotope effects on alpha-glucosidase

The solvent kinetic isotope effects (SKIE) on the yeast alpha-glucosidase-catalyzed hydrolysis of p-nitrophenyl and methyl-d-glucopyranoside were measured at 25 degrees C. With p-nitrophenyl-D-glucopyranoside (pNPG), the dependence of k(cat)/K(m) on pH (pD) revealed an unusually large (for glycohydrolases) solvent isotope effect on the pL-independent second-order rate constant, (DOD)(k(cat)/K(m)), of 1.9 (+/-0.3). The two pK(a)s characterizing the pH profile were increased in D(2)O. The shift in pK(a2) of 0.6 units is typical of acids of comparable acidity (pK(a)=6.5), but the increase in pK(a1) (=5.7) of 0.1 unit in going from H(2)O to D(2)O is unusually small. The initial velocities show substrate inhibition (K(is)/K(m) approximately 200) with a small solvent isotope effect on the inhibition constant [(DOD)K(is)=1.1 (+/-0.2)]. The solvent equilibrium isotope effects on the K(is) for the competitive inhibitors D-glucose and alpha-methyl D-glucoside are somewhat higher [(DOD)K(i)=1.5 (+/-0.1)]. Methyl glucoside is much less reactive than pNPG, with k(cat) 230 times lower and k(cat)/K(m) 5 x 10(4) times lower. The solvent isotope effect on k(cat) for this substrate [=1.11 (+/-0. 02)] is lower than that for pNPG [=1.67 (+/-0.07)], consistent with more extensive proton transfer in the transition state for the deglucosylation step than for the glucosylation step.     

Identification of critical steps governing the two-component alkanesulfonate monooxygenase catalytic mechanism

The alkanesulfonate monooxygenase enzyme (SsuD) catalyzes the oxygenolytic cleavage of a carbon-sulfur bond from sulfonated substrates. A mechanism involving acid-base catalysis has been proposed for the desulfonation mechanism by SsuD. In the proposed mechanism, base catalysis is involved in abstracting a proton from the alkane peroxyflavin intermediate, while acid catalysis is needed for the protonation of the FMNO(-) intermediate. The pH profiles of k(cat) indicate that catalysis by SsuD requires a group with a pK(a) of 6.6 ± 0.2 to be deprotonated and a second group with a pK(a) of 9.5 ± 0.1 to be protonated. The upper pK(a) value was not present in the pH profiles of k(cat)/K(m). Several conserved amino acid residues (His228, His11, His333, Cys54, and Arg226) have been identified as having potential catalytic importance due to the similar spatial arrangements with close structural and functional relatives of SsuD. Substitutions to these amino acid residues were generated, and the pH dependencies were evaluated and compared to wild-type SsuD. Although a histidine residue was previously proposed to be the active site base, the His variants possessed similar steady-state kinetic parameters as wild-type SsuD. Interestingly, R226A and R226K SsuD variants possessed undetectable activity, and there was no detectable formation of the C4a-(hydro)peroxyflavin intermediate for the Arg226 SsuD variants. Guanidinium rescue with the R226A SsuD variant resulted in the recovery of 1.5% of the wild-type SsuD k(cat) value. These results implicate Arg226 playing a critical role in catalysis and provide essential insights into the mechanistic steps that guide the SsuD desulfonation process.     

pH and kinetic isotope effects on sarcosine oxidation by N-methyltryptophan oxidase

N-Methyltryptophan oxidase (MTOX), a flavoenzyme from Escherichia coli, catalyzes the oxidative demethylation of secondary amino acids such as N-methyltryptophan or N-methylglycine (sarcosine). MTOX is one of several flavin-dependent amine oxidases whose chemical mechanism is still debated. The kinetic properties of MTOX with the slow substrate sarcosine were determined. Initial rate data are well-described by the equation for a ping-pong kinetic mechanism, in that the V/K(O)()2 value is independent of the sarcosine concentration at all accessible concentrations of oxygen. The k(cat)/K(sarc) pH profile is bell-shaped, with pK(a) values of 8.8 and about 10; the latter value matches the pK(a) value of the substrate nitrogen. The k(cat) pH profile exhibits a single pK(a) value of 9.1 for a group that must be unprotonated for catalysis. There is no significant solvent isotope effect on the k(cat)/K(sarc) value. With N-methyl-(2)H(3)-glycine as the substrate, there is a pH-independent kinetic isotope effect on k(cat), k(cat)/K(sarc), and the rate constant for flavin reduction, with an average value of 7.2. Stopped-flow spectroscopy with both the protiated and deuterated substrate failed to detect any intermediates between the enzyme-substrate complex and the fully reduced enzyme. These results are used to evaluate proposed chemical mechanisms.     

Substrate specificity of copper-containing plant amine oxidases

The steady-state kinetic parameters of the amine oxidases purified from Lathyrus cicera (LCAO) and Pisum sativum (PSAO) seedling were measured on a series of common substrates, previously tested on bovine serum amine oxidase (BSAO). LCAO, as PSAO, was substantially more reactive than BSAO with aliphatic diamines and histamine. The k(cat) and k(cat)/Km for putrescine were four and six order of magnitude higher, respectively. Differences were smaller with some aromatic monoamines. The plot of k(cat) versus hydrogen ions concentration produced bell-shaped curves, the maximum of which was substrate dependent, shifting from neutral pH with putrescine to alkaline pH with phenylethylamine and benzylamine. The latter substrates made the site more hydrophobic and increased the pK(a) of both enzyme-substrate and enzyme-product adducts. The plot of k(cat)/Km versus hydrogen ion concentration produced approximately parallel bell-shaped curves. Similar pK(a) couples were obtained from the latter curves, in agreement with the assignment as free enzyme and free substrate pK(a). The limited pH dependence of kinetic parameters suggests a predominance of hydrophobic interactions.     

Proton inventories constitute reliable tools in investigating enzymatic mechanisms: application on a novel thermo-stable extracellular protease from a halo-alkalophilic Bacillus sp

A novel protease designated protease-A-17N-1, was purified from the halo-alkalophilic Bacillus sp. 17N-1, and found active in media containing dithiothreitol and EDTAK(2). This enzyme maintained significant activity from pH 6.00 to 9.00, showed optimum k(cat)/K(m) value at pH 7.50 and 33 degrees C. It was observed that only specific inhibitors of cysteine proteinases inhibited its activity. The pH-(k(cat)/K(m)) profile of protease-A-17N-1 was described by three pK(a)s in the acid limb, and one in the alkaline limb. Both are more likely due t3o the protonic dissociation of an acidic residue, and the development and subsequent deprotonation of an ion-pair, respectively, in its catalytic site, characteristic for cysteine proteinases. Moreover, both the obtained estimates of rate constant k(1) and the ratio k(2)/k(-1) at 25 degrees C, from the temperature-(k(cat)/K(m)) profile of protease-A-17N-1, were found similar to those estimated from the proton inventories of the same parameter, verifying the reliability of the latter methodology. Besides, the bowed-downward proton inventories of k(cat)/K(m), as well as the large inverse SIE observed for this parameter, in combination with its dependence versus temperature, were showed unambiguously that k(cat)/K(m) = k(1). Such results suggest that the novel enzyme is more likely to be a cysteine proteinase functioning via a general acid-base mechanism.     

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.