Stopped-flow cryoenzymological investigation of the pre-steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns by leucine aminopeptidase

Stopped-flow fluorescence experiments have been carried out to study the steady-state kinetics of hydrolysis of Leu-Gly-NHNH-Dns [Dns = 5-(dimethylamino)naphthalene-1-sulfonyl] by porcine kidney cytosol leucine aminopeptidase (LAP) in 50% v/v methanol/buffer solution at ambient temperature and the pre-steady-state kinetics of this reaction in the -35 to 0 degrees C temperature range. Experiments have been carried out on LAP species containing Mg(II), Mn(II), Cu(II), Ni(II), Zn(II), and no metal ion at the regulatory metal binding site. At ambient temperatures, the stopped-flow fluorescence changes observed on hydrolysis of the substrate have been used to measure the steady-state kinetic parameters kcat and KM. The results show that 50% v/v methanol lowers the values of kcat from 2- to 12-fold compared to the reactions in the absence of methanol for all of the metallo-LAP, but that the values of KM are essentially unaffected. The pre-steady-state reactions carried out under nonturnover conditions at -35 degrees C reveal a new relaxation for LAP species with Ni(II), Cu(II), and Zn(II) in the regulatory site. The value of kobsd for this relaxation reaches a plateau at high substrate concentrations, and the magnitude of its fluorescence change at a fixed concentration of substrate is proportional to the enzyme concentration. Thus, this relaxation corresponds to the production and decay of a new enzyme-substrate intermediate not observed at higher temperatures whose fluorescence differs from that of the succeeding intermediate that is normally seen above -26 degrees C.     

Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases

Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange. The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions. For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min. However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent. The kinetic parameters Kcat and KM have been determined for each of the active metallospecies. The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM. This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.     

Effect of metal ions on the fluorescence of leucine aminopeptidase and its dansyl-peptide substrates

The effect of Cu(II), Ni(II), Zn(II), Mg(II), and Mn(II) on the fluorescence of porcine kidney cytosol leucine aminopeptidase and three of its dansyl(Dns) peptide substrates, Leu-Gly-NHNH-Dns, Leu-Gly-NH(CH2)2NH-Dns, and Leu-Gly-NH(CH2)6NH-Dns, has been investigated. These five metal ions were chosen for study because each binds to the regulatory metal binding site of leucine aminopeptidase. Since the binding is relatively weak, kinetic studies of the different metalloderivatives of the enzyme are normally carried out in the presence of large molar excesses of these metal ions that can potentially affect both the enzyme and substrate. The fluorescence of all of the dansyl-peptides, as well as several other dansyl species, is quenched by Ni(II) and Cu(II), but not by Mg(II), Mn(II), or Zn(II). The absorption spectra of these dansyl substrates are also perturbed by Ni(II) and Cu(II). The rate at which maximal quenching for some dansyl species is attained after mixing with Ni(II) and Cu(II) is slow and the quenching is reversed on addition of EDTA. These results indicate that the quenching is the result of complex formation between the fluorophores and these metal ions. The association constants for the metal complexes have been determined from Stern-Volmer plots. In addition to complex formation, Ni(II) and Cu(II) cause the degradation of Leu-Gly-NHNH-Dns through a two step mechanism involving loss of dansic acid. Ni(II) and Cu(II) also partially quench the fluorescence of leucine aminopeptidase through contact with its surface accessible Trp residues. These observations indicate that care must be taken in stopped flow fluorescence studies of reactions between this enzyme and its dansyl substrates to avoid adverse effects brought about by Ni(II) and Cu(II).     

One- and two-metal ion catalysis: global single-turnover kinetic analysis of the PvuII endonuclease mechanism

Ester hydrolysis is one of the most ubiquitous reactions in biochemistry. Many of these reactions rely on metal ions for various mechanistic steps. A large number of metal-dependent nucleases have been crystallized with two metal ions in their active sites. In spite of an ongoing discussion about the roles of these metal ions in nucleic acid hydrolysis, there are very few studies which examine this issue using the native cofactor Mg(II) and global fitting of reaction progress curves. As part of a comprehensive study of the representative homodimeric PvuII endonuclease, we have collected single-turnover DNA cleavage data as a function of Mg(II) concentration and globally fit these data to a number of models which test various aspects of the metallonuclease mechanism. DNA association rate constants are approximately 100-fold higher in the presence of the catalytically nonsupportive Ca(II) versus the native cofactor Mg(II), highlighting an interesting cofactor difference. A pathway in which metal ions bind prior to DNA is kinetically favored. The data fit well to a model in which both one and two metal ions per active site (EM(2)S and EM(4)S, respectively) support cleavage. Interestingly, the cleavage rate for EM(2)S is approximately 100-fold slower than that displayed by EM(4)S. Collectively, these data indicate that for the PvuII system, catalysis involving one metal ion per active site can indeed occur, but that a more efficient two-metal ion mechanism can be operative under saturating metal ion (in vitro) conditions.     

Reversible inhibition of intestinal alkaline phosphatase by inositol hexaphosphate and its Cu(II) coordinate complexes

The influence of inositol hexakisphosphate (IHP) and its cupric ion chelate complexes on alkaline phosphatase (APase) catalysis of p-nitrophenyl phosphate hydrolysis at pH 7.2 has been determined. Both IHP and (IHP-Cu) complexes, but not Cu(II) alone, are effective inhibitors of the enzyme and are of the strictly competitive type with Ki values in the microM range. Without added inhibitors present, the kinetic parameters are kcat 5.7 x 10(3) min(-1); and KM, 18 microM. In the presence of 62 microM IHP, kcat was essentially unchanged with an apparent KM of 68 microM giving a Ki of 22 microM. In the presence of an (IHP-Cu) complex (62 microM IHP, 128 microM Cu(II], the apparent KM was 55 microM and Ki was 30 microM. At a ratio of Cu(II):IHP of 6.0 (372:62 microM) the apparent KM was 30 microM and Ki was 94 microM. The inhibitory effect of (IHP-Cu) complexes thus decreases as the IHP binding sites for cupric ions become saturated. A high ionic strength environment markedly reduces the inhibitory effect of IHP. Previous studies have also shown that rates of APase inactivation by (IHP-Cu) complexes are also ionic strength sensitive [1]. The inhibition of APase activity by either IHP or its coordinate complexes with cupric ions is evidence for their interaction at the enzyme's catalytic sites. Such results thus provide support for an essential element of the mechanism previously suggested for the reversible inactivation (as opposed to inhibition) of APase by (IHP-Cu) chelate complexes, viz., that it may be due to a metal ion exchange reaction leading to the formation of a Cu(II)-substituted enzyme.     

Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interdependency of the binding subsites in subtilisin.

Subtilisins are endopeptidases with an extended binding cleft comprising at least eight subsites, and kinetic studies have revealed that subsites distant from the scissile bond are important in determining the substrate preference of the enzymes. With the subtilisin enzyme Savinase, the interdependency of the individual Sn-Pn interactions has been investigated. It was found that the contributions from each subsite interaction to kcat/KM are not always additive. Such interdependency was also observed between subsites which are remote from each other. With a series of substrates covering S6 to S'4 of Savinase, it was observed that favorable amino acids in P1 or, more significantly, P4 of the substrate shield adverse effects of less favorable amino acids at other positions. Thus, an upper limit of kcat/KM was observed, suggesting a limit on the amount of substrate interaction energy which can be converted into transition-state stabilization. Furthermore, with substrates in which all positions had been optimized, an upper limit of kcat/KM (approximately 2 x 10(9) min-1 M-1) was seen, both for a substrate with a high kcat and for one with a low KM. These results emphasize that the design of optimal substrates or substrate-derived inhibitors for endopeptidases preferably should be based on subsite mappings where interdependent substrate-subsite interactions have been eliminated.     

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.     

One is enough: insights into the two-metal ion nuclease mechanism from global analysis and computational studies

The mechanistic details of metallonuclease reactions, typically supported by Mg(II), have a long and contentious history. Two-metal ion mechanisms have enjoyed much favor, based largely in the multitude of X-ray crystal structures of these enzymes with more than one metal ion per active site. Most recently, this mechanism has come under challenge. Reviewed herein are the applications of different experimental strategies that collectively support a mechanism in which only one metal ion is necessary for nucleic acid hydrolysis. Based on global kinetic analysis, analysis of reactions in which the nonsupportive Ca(ii) is added, and a number of computational approaches, secondary sites are proposed to either be occupied by activity-modulating metal ions or occupied in turn by a single metal that changes position during the course of the reaction.     

Sequence specificity of human skin fibroblast collagenase. Evidence for the role of collagen structure in determining the collagenase cleavage site

The sequence specificity of human skin fibroblast collagenase has been investigated by measuring the rate of hydrolysis of 16 synthetic octapeptides covering the P4 through P4' subsites of the substrate. The choice of peptides was patterned after potential collagenase cleavage sites (those containing either the Gly-Leu-Ala or Gly-Ile-Ala sequences) found in types I, II, and III collagens. The initial rate of hydrolysis of the P1-P1' bond of each peptide has been measured by quantitating the concentration of amino groups produced upon cleavage after reaction with fluorescamine. The reactions have been carried out under first-order conditions ([S] much less than KM) and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P3 (Pro, Ala, Leu, or Asn), P2 (Gln, Leu, Hyp, Arg, Asp, or Val), P1' (Ile or Leu), and P4' (Gln, Thr, His, Ala, or Pro) all influence the hydrolysis rates. However, the differences in the relative rates observed for these octapeptides cannot in themselves explain why fibroblast collagenase hydrolyzes only the Gly-Leu and Gly-Ile bonds found at the cleavage site of native collagens. This supports the notion that the local structure of collagen is important in determining the location of the mammalian collagenase cleavage site.     

Characterizing metalloendonuclease mixed metal complexes by global kinetic analysis

To test the role of a secondary metal ion in a two metal ion metallonuclease mechanism, some groups have introduced a nonsupportive metal ion [usually Ca(II)] in cleavage reactions. Stimulation of Mg(II)- or Mn(II)-supported activity has been taken as evidence that the second metal ion is regulatory. However, this activity has yet to be dissected to determine what processes and species contribute to this observation. Here, we test global kinetic analysis as an approach to this problem. Taking advantage of the various binding and cleavage constants established for PvuII endonuclease, we apply cleavage data obtained under a range of Mg(II) and Ca(II) concentrations to a number of kinetic models which specify A and B sites for both metal ions and various active species. The data are best fit and simulated with models which feature Ca(II) being held more strongly in the B (or secondary) site. This mixed metal enzyme species is the only one which forms appreciably and exhibits a cleavage rate constant similar to that observed when there is only one Mg(II) per active site (approximately 0.01 s^?1). Thus, in the case of PvuII endonuclease, Ca(II) does not stimulate cleavage. However, a simulated increase in activity at moderate Ca(II) concentrations can be rationalized with a cleavage rate constant for the mixed species similar to that when two Mg(II) ions are present in the active site. This provides an important insight into the underlying basis for the Ca(II)-stimulated activity observed for some metallonucleases that is not accessible by any other means.     

Substrate specificity and kinetic characteristics of angiotensin converting enzyme

Furanacryloyl-Phe-Gly-Gly has been shown to be a convenient substrate for angiotensin converting enzyme (dipeptidyl carboxypeptidase, EC 3.4.15.1). A detailed kinetic analysis of the hydrolysis of this substrate indicates normal Michaelis-Menten behavior with kcat = 19000 min-1 and KM = 3.0 x 10(-4) M determined at pH 7.5, 25 degrees C. The enzyme is inhibited by phosphate and activated by chloride; maximal activity is observed with 300 mM NaCl. In the absence of added zinc, activity is lost rapidly below pH 7.5 due to spontaneous dissociation of the metal, but in the presence of zinc, the enzyme remains fully active to about pH 6. The pH-rate profile indicates two groups on the enzyme with apparent pK values of 5.6 and 8.4. The substrate specificity of the enzyme has been examined in terms of the fundamental specificity quantity kcat/KM as well as the separate constants by using a series of furanacryloyl-tripeptides. The activity toward furanacryloyl-Phe-Gly-Gly has been compared with that toward the physiological substrates angiotensin I and bradykinin.     

Stopped-flow kinetic studies of metal ion dissociation or exchange in a tryptophan-containing parvalbumin

The rates of dissociation of 2 equiv of various metal ions [Ca(II), Cd(II), Pr(III), Nd(III), Sm(III), Eu(III), Gd(III), Tb(III), Dy(III), Ho(III), Er(III), Yb(III), and Lu(III)] from the primary CD and EF metal ion binding sites of parvalbumin (isotype pI = 4.75) from codfish (Gadus callarius L) were measured by stopped-flow techniques. The removal or replacement of metal ions was monitored by changes in sensitized Tb(III) luminescence or in intrinsic protein tryptophan fluorescence as quenching ions [Eu(III) or Yb(III)] were bound or removed or as the apoprotein was formed. In experiments wherein the bound metal ions were removed by mixing the parvalbumin with an excess of 1,2-diaminocyclohexanetetraacetic acid (DCTA), the kinetic traces were best fit by a double exponential with koff rate constants of 1.07 and 5.91 s-1 for Ca(II), 1.54 and 10.5 s-1 for Cd(II), and approximately 0.05 and approximately 0.5 s-1 for all of the trivalent lanthanide ions. In experiments wherein the bound metal ions were exchanged with an excess of a different metal ion, pseudo-first-order rate constants were proportional to the concentration of excess attacking metal ion for both the fast and slow processes in most experiments. In these cases, extrapolation of the rate constants to zero concentration of attacking metal ion gave values which agree well with the DCTA scavenging results. This finding demonstrates that the off rate constants do not depend on the occupancy of the neighboring site and therefore implies that there is no significant cooperativity in metal ion binding between the two sites in parvalbumin.     

Activation of hypoxanthine/guanine phosphoribosyltransferase from yeast by divalent zinc and nickel ions

We have observed previously that the reactions catalyzed by hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) are activated by Mg(II), Mn(II), and Co(II), and we have defined the mechanism by which these activations proceed [Biochemistry 22, 3419-3424 (1983)]. A more extensive survey of the kinds of metal ions that will activate the HGPRTase catalysis now has been completed through the use of an HPLC assay procedure. Although Fe(II) and Ca(II) are unable to activate this reaction, a significant activation was achieved with the addition of spectroscopically pure Zn(II) to the assay solution. In addition some IMP synthesis resulted from the addition of Ni(II) to the assay mixture. Both the Zn(II) and Ni(II) kinetic effects on HGPRTase over a limited metal ion concentration range have been analyzed through the use of curve-fitting exercises. These results, in addition to the similar pH profiles for the activations by Mg(II), Mn(II), Co(II), and Zn(II), suggest that all of these metal ions activate the HGPRTase-catalyzed synthesis of IMP by way of the same mechanism [model II as defined by London and Steck, Biochemistry 8, 1767-1779 (1969)], during which two divalent ions bind to the HGPRTase active site per molecule of PRibPP.     

Characterization of the catalytic defect in the dysthrombin, Thrombin Quick

The dysthrombin, Thrombin Quick, is chromatographically separable into two components designated Thrombin Quick I and Thrombin Quick II. Thrombin Quick II lacks observable catalytic activity toward thrombin substrates. The steady-state kinetics of hydrolysis of benzoylarginine ethyl ester and Tos-Gly-Pro-Arg-p-nitroanilide by Thrombin Quick I are equivalent to those of thrombin. These results, in addition to binding studies with the active site titrant N2-(5-dimethylaminonaphthalene-1-sulfonyl)arginine N-(3-ethyl-1,5-pentanediyl)amide, indicate that binding interactions at the catalytic site of Thrombin Quick I are unaltered. Thrombin Quick I is inhibited by anti-thrombin III at the same rate as thrombin. Steady-state kinetic parameters for the release of fibrinopeptide A indicate defects in both kcat and Km for Thrombin Quick I with kcat/Km equal to 0.012 of the value for thrombin, corresponding to the relative fibrinogen clotting activity of 0.013. The results are interpreted as indicating a defect in Thrombin Quick I at a binding site, external to the catalytic site, which is essential for determining specificity toward fibrinogen. The defect in kcat may result secondarily from small perturbations in the steric relationship of the catalytic triad residues. The rate of hydrolysis by Thrombin Quick I of the protein substrates bovine prothrombin and bovine protein C (in the absence of cofactors) is about one-third of that observed for thrombin, indicating that hydrolysis of these substrates by thrombin involves different specificity determinants than does the hydrolysis of fibrinogen.     

Observation of a chloride-dependent intermediate during catalysis by angiotensin converting enzyme using radiationless energy transfer

Stopped-flow radiationless energy-transfer kinetics have been used to examine the effects of chloride on the hydrolysis of Dns-Lys-Phe-Ala-Arg by angiotensin converting enzyme. The kinetic constants for hydrolysis at pH 7.5 and 22 degrees C in the presence of 300 mM sodium chloride were KM = 28 microM and kcat = 110 s-1, and in its absence, KM = 240 microM and kcat = 68 s-1. The apparent binding constant for chloride was 4 mM, and the extent of chloride activation in terms of kcat/KM was 14-fold. The effects of chloride on the pre-steady-state were examined at 2 degrees C. In the presence of chloride, two distinct enzyme-substrate complexes were observed, suggesting multiple steps in substrate binding. The initial complex was formed during the mixing period (kobsd greater than 200 s-1) while the second complex was formed much more slowly (kobsd = 40 s-1 when [S] = 5 microM and [NaCl] = 150 mM). Strikingly, in the absence of chloride, only a single, rapidly formed enzyme-substrate complex was observed. These results are consistent with a nonessential activator kinetic mechanism in which the slow step reflects conversion of an initially formed complex, (E X Cl- X S)1, to a more tightly bound complex, (E X Cl- X S)2.     

Reactivity of bovine blood coagulation factor IXa beta, factor Xa beta, and factor XIa toward fluorogenic peptides containing the activation site sequences of bovine factor IX and factor X

The published activation site sequences of bovine factors IX and X have been utilized to synthesize a number of peptides specifically designed respectively as substrates for bovine factors XIa and IXa beta. The substrates contain a fluorophore (2-aminobenzoyl group, Abz) and a quenching group (4-nitrobenzylamide, Nba) that are separated upon enzymatic hydrolysis with a resultant increase in fluorescence that was utilized to measure hydrolysis rates. Factor XIa cleaved all of the peptides bearing factor IX activation site sequences with Abz-Glu-Phe-Ser-Arg-Val-Val-Gly-Nba having the highest kcat/KM value. The kinetic behavior of factor XIa toward the synthetic peptide substrate indicates that it has a minimal extended substrate recognition site at least five residues long spanning S4 to S1' and has favorable interactions over seven subsites. The hexapeptide Abz-Glu-Phe-Ser-Arg-Val-Val-Nba was the most specific factor XIa substrate and was not hydrolyzed by factors IXa beta or Xa beta or thrombin. Factor IXa beta failed to hydrolyze any of the synthetic peptides bearing the activation site sequence of factor X. This enzyme slowly cleaved four hexa- and heptapeptide substrates with factor IX activation site sequences extending from P4 or P3 to P3'. Factor Xa beta poorly hydrolyzed all but one of the factor XIa substrates and failed to cleave any of the factor IXa beta substrates. Thrombin failed to hydrolyze any of the peptides examined while trypsin, as expected, was highly reactive and not very specific. Phospholipids had no effect on the reactivity of either factors IXa beta or Xa beta toward synthetic substrates. Both factor IXa beta and Xa beta cleaved the peptide substrates at similar rates to their natural substrates under comparable conditions. However the rates were substantially lower than optimum activation rates observed in the presence of Ca2+, phospholipids, and protein cofactors. In the future, it may be useful to investigate synthetic substrates that can bind to phospholipid vesicles in the same manner as the natural substrates for factors IXa beta and Xa beta.     

Hydrolysis of glycine-containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross-links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine-rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 +/- 9 min(-1) x mmol(-1) x L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation-mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG(cleavage site)A being especially sensitive to hydrolysis. Both positions P2 and P'2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P'3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.     

Mechanism of GlvA from Bacillus subtilis: a detailed kinetic analysis of a 6-phospho-alpha-glucosidase from glycoside hydrolase family 4

GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis assigned to glycoside hydrolase family 4, catalyzes the hydrolysis of maltose 6'-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium, consistent with C-H bond cleavage at each center being partially rate-limiting. Kinetic parameters were also determined for 1-[2H]-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Br?nsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase in glycoside hydrolase family 4 (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured and the observation of very similar kcat values for all substrates, including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site, yielding the product sugar-6-phosphate in the same anomeric form in the two cases.     

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.