Sturgeon proopiomelanocortin has a remnant of gamma-melanotropin

A thioesterase I gene was recloned and sequenced from Escherichia coli strain JM109. The overexpressed, matured enzyme from JM109 was purified to homogeneity. The enzyme showed broad hydrolytic activity toward three kinds of substrates including acyl-CoAs, esters, and amino acid derivatives. The enzyme had a kcat/Km value of 0.363 s-1 microM-1, for a typical thioesterase I substrate, palmitoyl-CoA. The arylesterase activity of the enzyme was observed by its ability to hydrolyze several aromatic esters including alpha-naphthyl acetate, alpha-naphthyl butyrate, phenyl acetate, benzyl acetate, and eight p-nitrophenyl esters. In kinetic studies a chymotrypsin-like substrate (an amino acid derivative), N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (L-NBPNPE), was the best substrate for the enzyme with a catalytic efficiency (kcat/Km) of 4.00 s-1 microM-1, which was 23 times higher than that of the enantiomer D-NBPNPE (0.171 s-1 microM-1). It was concluded that the thioesterase I of E. coli had arylesterase activity and it possessed stereospecificity for protease substrates.     

The bifunctional enzyme leukotriene-A4 hydrolase is an arginine aminopeptidase of high efficiency and specificity

Leukotriene-A4 hydrolase (EC 3.3.2.6) cleaved the NH2-terminal amino acid from several tripeptides, typified by arginyl-glycyl-aspartic acid, arginyl-glycyl-glycine, and arginyl-histidyl-phenylalanine, with catalytic efficiencies (kcat/Km) > or = 1 x 10(6) M-1 s-1. This exceeds by 10-fold the kcat/Km for its lipid substrate leukotriene A4. Catalytic efficiency declined for dipeptides which had kcat/Km ratios 10-100-fold lower than tripeptides. Tetrapeptides and pentapeptides were even poorer substrates with catalytic efficiencies below 10(3) M-1 s-1. The enzyme preferentially hydrolyzed tripeptide substrates and single amino acid p-nitroanilides with L-arginine at the NH2 terminus. Peptides with proline at the second position were not hydrolyzed, suggesting a requirement for an N-hydrogen at the peptide bond cleaved. Peptides with a blocked NH2 terminus were not hydrolyzed. The specificity constant (kcat/Km) was optimal at pH 7.2 with pK values at 6.8 and 7.9; binding was maximal at pH 8.0. Serum albumins activated the peptidase, increasing tripeptide affinities (Km) by 3-10-fold and specificities (kcat/Km) by 4-13-fold. Two known inhibitors of arginine peptidases, arphamenine A and B, inhibited hydrolysis of L-arginine p-nitroanilide with dissociation constants = 2.0 and 2.5 microM, respectively. Although the primary role of LTA4 hydrolase is widely regarded as the conversion of the lipid substrate leukotriene A4 into the inflammatory lipid mediator leukotriene B4, our data are the first showing that tripeptides are "better" substrates. This is compatible with a biological role for the peptidase activity of the enzyme and may be relevant to the distribution of the enzyme in organs like the ileum, liver, lung, and brain. We present a model which accommodates the available data on the interaction of substrates and inhibitors with the enzyme. This model can account for overlap in the active site for hydrolysis of leukotriene A4 and peptide or p-nitroanilide substrates.     

Substituted glycals as probes of glycosidase mechanisms

D-Glucal and a series of substituted derivatives have been tested as substrates, inhibitors and inactivators of the Agrobacterium faecalis beta-glucosidase in order to probe structure/function relationships in this enzyme. D-Glucal is shown to be a substrate (kcat = 2.3 min-1, Km = 0.85 mM) undergoing hydration with stereospecific protonation from the alpha-face to yield 2-deoxy-beta-D-glucose. 1-Methyl-D-glucal surprisingly serves as only a poor substrate (kcat = 0.056 min-1, Km = 57 mM), also undergoing protonation from the alpha-face. 2-Fluoro-D-glucal, however is completely inert, as a result of inductive destabilisation of the oxocarbenium ion-like transition state for protonation, and functions only as a relatively weak (Ki = 24 mM) inhibitor. Similar behaviour was seen with almond beta-glucosidase and yeast alpha-glucosidase and for the interaction of 2-fluoro-D-galactal with Escherichia coli beta-galactosidase. A series of of alpha, beta-unsaturated glucal derivatives was also synthesised and tested as potential substrates, inhibitors or inactivators of A. faecalis beta-glucosidase. Of these only 1-nitro-D-glucal functioned as a time dependent, irreversible inactivator (ki = 0.011 min-1, Ki = 5.5 mM), presumably acting as a Michael acceptor. Electrospray mass spectrometric analysis revealed multiple labeling of the enzyme by this inactivator, lessening its usefulness as an affinity label. Less reactive Michael acceptor glycals which might have been more specific (1-cyano-, 2-cyano-, 1-carboxylic acid, 1-carboxylic acid methyl ester) unfortunately did not function as inactivators or substrates, only as relatively weak reversible inhibitors (Ki = 3-96 mM).     

Substrate specificity of honeydew melon protease D, a plant serine endopeptidase

The substrate specificity of honeydew melon (Cucumis melo var. inodorus Naud) protease D was studied by the use of synthetic substrates and oligopeptides derived from a protein hydrolyzate. The hydrolysis rates of succinyl-(L-Ala)1-3-p-nitroanilide (Suc-(Ala)1-3-pNA) the hydrolysis rate progressively rose in proportion to the increased chain length. Benzyloxycarbonyl-L-tyrosine p-nitrophenyl ester (Z-Tyr-ONp) and benzoyl-L-tyrosine ethyl ester (Bz-Tyr-OEt) were cleaved by honeydew melon protease D, but benzoyl-L-arginine p-nitroanilide (Bz-Arg-pNA), benzyloxycarbonyl-L-lysine p-nitrophenyl ester (Z-Lys-ONp) and tosyl-L-arginine methyl ester (Tos-Arg-OMe) were not hydrolyzed. Contrary to the results obtained by using synthetic substrates, the carboxyl sides of charged amino acid residues were preferentially cleaved by the enzyme in the oligopeptide substrates. The substrates that had charged or polar amino acids at P2 positions were not cleaved. On the other hand, the non-polar amino acid or proline at P2 were favored for hydrolysis. The information concerning the subsite of protease D was obtained and is useful for synthesis of a good substrate. As it is distinct from molecular mass, the substrate specificity of honeydew melon protease D is most analogous to cucumisin [EC 3.4.21.25] among serine proteases from cucurbitaceous plants.     

Ricin A-chain: kinetics, mechanism, and RNA stem-loop inhibitors

Ricin A-chain (RTA) catalyzes the depurination of a single adenine at position 4324 of 28S rRNA in a N-ribohydrolase reaction. The mechanism and specificity for RTA are examined using RNA stem-loop structures of 10-18 nucleotides which contain the required substrate motif, a GAGA tetraloop. At the optimal pH near 4.0, the preferred substrate is a 14-base stem-loop RNA which is hydrolyzed at 219 min-1 with a kcat/Km of 4.5 x 10(5) M-1 s-1 under conditions of steady-state catalysis. Smaller or larger stem-loop RNAs have lower kcat values, but all have Km values of approximately 5 microM. Both the 10- and 18-base substrates have kcat/Km near 10(4) M-1 s-1. Covalent cross-linking of the stem has a small effect on the kinetic parameters. Stem-loop DNA (10 bases) of the same sequence is also a substrate with a kcat/Km of 0.1 that for RNA. Chemical mechanisms for enzymatic RNA depurination reactions include leaving group activation, stabilization of a ribooxocarbenium transition state, a covalent enzyme-ribosyl intermediate, and ionization of the 2'-hydroxyl. A stem-loop RNA with p-nitrophenyl O-riboside at the depurination site is not a substrate, but binds tightly to the enzyme (Ki = 0.34 microM), consistent with a catalytic mechanism of leaving group activation. The substrate activity of stem-loop DNA eliminates ionization of the 2'-hydroxyl as a mechanism. Incorporation of the C-riboside formycin A at the depurination site provides an increased pKa of the adenine analogue at N7. Binding of this analogue (Ki = 9.4 microM) is weaker than substrate which indicates that the altered pKa at this position is not an important feature of transition state recognition. Stem-loop RNA with phenyliminoribitol at the depurination site increases the affinity substantially (Ki = 0.18 microM). The results are consistent with catalysis occurring by leaving group protonation at ring position(s) other than N7 leading to a ribooxocarbenium ion transition state. Small stem-loop RNAs have been identified with substrate activity within an order of magnitude of that reported for intact ribosomes.     

Mechanism of pancreatic lipase action. 1. Interfacial activation of pancreatic lipase

Hydrolysis of dissolved p-nitrophenyl acetate by pancreatic lipase follows the classical acyl enzyme pathway already proposed for other esterases. Kinetic parameters of the hydrolysis have been determined. The turnover rate of the reaction is many orders of magnitude slower than that for the natural emulsified substrates. Nevertheless, several arguments are in favor of the specificity of this hydrolysis: (1) triacetin, which resembles the usual substrates for the enzyme, is also hydrolyzed very slowly in solution; (2) dissolved triacetin and tripropionin are competitive inhibitors for the p-nitrophenyl acetate hydrolysis; (3) the same chemical structural features which are required in the case of emulsified substrates are also necessary to promote hydrolysis of dissolved p-nitrophenyl esters. This suggests that the same active site (or part of the same active site) is responsible for hydrolysis of both p-nitrophenyy acetate and specific emulsified substrates. Since deacylation is the rate-limiting step in the catalysis of p-nitrophenyl acetate, the intermediate acetyl enzyme can be isolated by trapping it at pH 5.0. Kinetic competence of this intermediate has been demonstrated. Hydrolysis by pancreatic lipase of dissolved monomeric p-nitrophenyl acetate and triacetin is considerably enhanced (100- to 500-fold) by various interfaces. This suggests that at least the deacylation step, which is rate limiting in absence of interface, is accelerated by the presence of inert interfaces. Siliconized glass beads were directly shown to accelerate the deacylation of isolated [3H]acetyl lipase by at least a hundred times. This step does not directly involve the ester substrate.Thus, it is suggested that a part of the activation of lipase at interfaces may be due to a conformational change resulting from adsorption.     

Observations on the relationship of parathyroid hormone and calcitonin to plasma and liver phosphate

1. The action of two active forms of bovine trypsin (alpha and beta-trypsin) on a series of specific methyl ester substrates of general formula: N-acetyl-(glycyl)n-L-lysine methyl ester (n = 0, 1, 2) and N2-benzoyl-L-arginine ethyl ester have been investigated. With the L-lysine methyl esters the catalytic rate constant for hydrolysis (kcat) was found to be significantly lower for alpha-trypsin than for beta-trypsin, whereas with N2-benzoyl-L-arginine ethyl ester there was no significant difference for the two enzymes. 2. By measurement of the kinetic constants (kcat and Km) in the presence of a nucleophile, which competes with water in the deacylation process, it has been shown that, in common with the specific ester substrates of trypsin, the rate-determining step for the extended L-lysine methyl esters is decaylation of the enzyme. 3. It has been found that by extending the aminoacyl group of N-acetyl-L-lysine methyl ester by one glycine residue (n = 1), a greatly enhanced deacylation rate constant is observed for both alpha and beta-trypsin. The higher rate constants were maintained at the higher levels by the addition of a further glycine residue (n = 2). These results have been interpreted in terms of the 'induced fit' hypothesis the substrates binding to an enzyme subsite adjacent to the active site. 4. The beta-trypsin-catalysed hydrolysis of the L-lysine substrates was investigated over a range of temperature (15--35 degrees C). The Arrhenius law was obeyed, within experimental error, by all three substrates allowing the estimation of the thermodynamic function of activation (delta S not equal to and deltaH note equal to) for the deacylation reactions. The significantly higher values of deltaS not equal to and deltaH not equal to obtained for the two extended substrates are interpreted in terms of additional hydrogen bonding between the longer aminoacyl chains and the enzyme molecule. The results are compared with those for non-extended specific substrates, which have a possible hydrophobic interaction with the enzyme surface.     

Purification and characterisation of an extracellular beta-glucosidase with transglycosylation and exo-glucosidase activities from Fusarium oxysporum

An extracellular beta-glucosidase from Fusarium oxysporum was purified to homogeneity by gel-filtration and ion-exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0-6.0 and at 60 degrees C. It hydrolysed 1-->4-linked aryl-beta-glucosides and 1-->4-linked, 1-->3-linked and 1-->6-linked beta-glucosides. The apparent Km and kcat values for p-nitrophenyl beta-D-glucopyranoside (4-NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min-1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 microM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2-fold at 0.5 M) while methanol increased the activity by 1.4-fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello-oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, beta-glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.     

Kinetics of the hydrolysis of synthetic substrates by horse urinary kallikrein and trypsin

The kinetic constants for horse urinary kallikrein and trypsin hydrolysis of BAEE, TAME, bradykinin methyl ester and bradykinyl-Ser-Val-Gin-Val-Ser were determined. The values of the ratio kcat/Km show that (1) kallikrein is catalytically less efficient than trypsin for all the substrates (2) the three esters are equally good substrates for trypsin while horse urinary kallikrein is 100-fold more effective on bradykinin methyl ester than on the other substrates (3) for both enzymes the ester of bradykinin is a better substrate than the tetradecapeptide.     

Kinetic analysis of papaya proteinase omega

Papaya proteinase omega (pp omega) has been purified from dried latex both by immunoaffinity and traditional methods. Kinetic analysis revealed that (1), the pp omega-catalysed hydrolysis of N-benzoyl-L-arginine p-nitroanilide (BApNA) has a lower specificity (kcat/Km) than the same reaction catalysed by papain; (2), the pp omega-catalysed hydrolysis of a tripeptide substrate having phenylalanine at the second position (S2-site) showed a more similar specificity to that catalysed by papain; (3), the significant difference between the two enzymes is that steady state kinetics with both L-BApNA and a tripeptide enables the identification in pp omega of other ionizations affecting binding. The active sites of papain and pp omega can therefore be distinguished by pH-dependence of kcat/Km.     

Osteogenic protein-1 regulates insulin-like growth factor-I (IGF-I), IGF-II, and IGF-binding protein-5 (IGFBP-5) gene expression in fetal rat calvaria cells by different mechanisms

Agrobacterium tumefaciens beta-glucosidase, Cbg1 was extensively characterised and found to be a retaining aryl-glucosidase and an aryl-xylosidase. Cbg1s specificity for p-nitrophenyl beta-d-xylopyranoside was 73% that for p-nitrophenyl beta-d-glucopyranoside when measured by the ratio kcat/Km. The enzyme also hydrolysed p-nitrophenyl beta-d-fucopyranoside, and p-nitrophenyl beta-d-galactopyranoside with moderate efficiency. The enzyme released only terminal glucose from p-nitrophenyl beta-cellobioside and had a 20 000-fold preference for its natural substrate coniferin over cellobiose as indicated by the ratio kcat/Km. The enzyme was activated in the presence of 20 mM 1-propanol, 1-butanol, 1-pentanol, 1-hexanol, 1-heptanol, and 1-octanol. In the case of butanol this activation was investigated and shown to be due to transglycosylation activity with over 80% of p-nitrophenyl beta-d-glucopyranoside being converted to 1-butyl beta-d-glucopyranoside in the presence of Cbg1 and 100 mM 1-butanol.     

Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support

The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media. The free enzyme was mixed with bovine serum albumin and lyophilized. Immobilization was on porous polypropylene. Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined. The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively. Both the free and immobilized lipases followed Michaelis-Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture. For pNPP, the activation factor upon immobilization came mainly from a reduction in Km)app while kcat was increased for pNPA.     

Mechanisms of cellulases and xylanases: a detailed kinetic study of the exo-beta-1,4-glycanase from Cellulomonas fimi

The exoglucanase/xylanase from Cellulomonas fimi (Cex) has been subjected to a detailed kinetic investigation with a range of aryl beta-D-glycoside substrates. This enzyme hydrolyzes its substrates with net retention of anomeric configuration, and thus it presumably follows a double-displacement mechanism. Values of kcat are found to be invariant with pH whereas kcat/Km is dependent upon two ionizations of pKa = 4.1 and 7.7. The substrate preference of the enzyme increases in the order glucosides < cellobiosides < xylobiosides, and kinetic studies with a range of aryl glucosides and cellobiosides have allowed construction of Broensted relationships for these substrate types. A strong dependence of both kcat (beta 1g = -1) and kcat/Km (beta 1g = -1) upon leaving group ability is observed for the glucosides, indicating that formation of the intermediate is rate-limiting. For the cellobiosides a biphasic, concave downward plot is seenj for kcat, indicating a change in rate-determining step across the series. Pre-steady-state kinetic experiments allowed construction of linear Broensted plots of log k2 and log (k2/Kd) for the cellobiosides of modest (beta 1g = -0.3) slope. These results are consistent with a double-displacement mechanism in which a glycosyl-enzyme intermediate is formed and hydrolyzed via oxocarbonium ion-like transition states. Secondary deuterium kinetic isotope effects and inactivation experiments provide further insight into transition-state structures and, in concert with beta 1g values, reveal that the presence of the distal sugar moiety in cellobiosides results in a less highly charged transition state.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificities of pepstatin-insensitive carboxyl proteinases from gram-negative bacteria

Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp. 101, and Xanthomonas carboxyl proteinase (XCP), isolated from Xanthomonas sp. T-22, are the first and second examples of unique carboxyl proteinases [EC 3.4.23.33] which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane. The substrate specificities of PCP and XCP were studied using a series of synthetic chromogenic peptide substrates with the general structure, P5-P4-P3-P2-Phe-Nph-P2'-P3' (P5, P4, P3, P2, P2', P3': a variety of amino acids, Nph is p-nitro-L-phenylalanine, and the Phe-Nph bond is cleaved). PCP and XCP were shown to hydrolyze a synthetic substrate, Lys-Pro-Ala-Leu-Phe-Nph-Arg-Leu, most effectively among 28 substrates. The kinetic parameters of this peptide for PCP were Km = 6.3 microM, Kcat = 51.4 s-1, and kcat/Km = 8.16 microM-1.s-1. The kinetic parameters for XCP were Km = 3.6 microM, kcat = 52.2 s-1, and kcat/Km = 14.5 microM-1.s-1. PCP showed a stricter substrate specificity than XCP. That is, the specificity constant (kcat/Km) of each substrate for PCP was in general < 0.5 microM-1.s-1, but was drastically improved by the replacement of Lys by Leu at the P2 position. On the other hand, XCP showed a less stringent substrate specificity, with most of the peptides exhibiting reasonable kcat/Km values (> 1.0 microM-1.s-1). Thus it was found that the substrate specificities of PCP and XCP differ considerably, in spite of the high similarity in their primary structures. In addition, tyrostatin was found to be a competitive inhibitor for XCP, with a Ki value of 2.1 nM, as well as for PCP (Ki = 2.6 nM).     

Kinetic properties of saquinavir-resistant mutants of human immunodeficiency virus type 1 protease and their implications in drug resistance in vivo

In order to study the basis of resistance of human immunodeficiency virus, type 1 (HIV-1), to HIV-1 protease inhibitor saquinavir, the catalytic and inhibition properties of the wild-type HIV-1 protease and three saquinavir resistant mutants, G48V, L90M, and G48V/L90M, were compared. The kinetic parameter kcat/Km was determined for these proteases using eight peptide substrates whose sequences were derived from the natural processing site sequences of HIV-1. The kcat/Km values were determined using conventional steady-state kinetics as well as initial velocities of mixed substrate cleavages under the condition where the substrate concentrations [S]o < Km. The independently determined kcat and Km values for some of the substrates confirmed the accuracy of the mixed-substrate method and also permitted the calculation in all cases of true rather than relative kcat/Km values. The Ki values were also determined. Using a previously described kinetic model [Tang, J., & Hartsuck, J. A. (1995) FEBS Lett. 367, 112-116], the relative processing activities of HIV-1 protease variants were estimated in the saquinavir concentration range of 0-10(-7) M. Although the protease activity of G48V, L90M, and G48V/L90M are only about 10, 7, and 3% of that of the wild-type HIV-1 protease in the absence of inhibitor, the resistance tendencies of the three mutants are clearly manifest by relatively less activity loss as inhibitor concentration becomes higher. Also, the ratios of the activities of the four protease species at certain saquinavir concentrations appear to correlate with the population ratios of the four protease species at different time points of clinical trials. This correlation suggests that the population ratio of the protease species is driven by in vivo saquinavir concentration, which appears to be in the range 10(-10)-10(-9) M during the clinical trials.     

Papain-catalyzed reactions at subzero temperatures

As a first step in the investigation of papain catalysis using subzero temperatures to detect, accumulate, and characterize enzyme-substrate intermediates, we have studied some potential cryosolvents and carried out preliminary intermediate trapping experiments. The effects of subzero temperatures and aqueous dimethyl sulfoxide solutions on the papain-catalyzed hydrolysis of Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester have been investigated in detail. At 0 degrees C, the value of kcat decreases with increasing dimethyl sulfoxide concentration, decreasing in proportion to the decreased water concentration; however, the value of Km increases exponentially. The effect on Km can be accounted for by a combination of both dielectric and competitive inhibition effects. The Arrhenius plot for the deacylation reaction in 7.65 M (60% v/v) dimethyl sulfoxide is linear over the temperature range 0 to -45 degrees C and extrapolates to a calculated value of kcat at 25 degrees C in excellent agreement with that obtained in the absence of organic solvent. The pH-rate profile is not substantially perturbed by the presence of 7.65 M dimethyl sulfoxide. At -45 degrees C and below, turnover occurs extremely slowly, and is essentially negligible, although acylation is still quite rapid. Consequently, the acyl enzyme, Na-carbobenzoxy-L-lysyl-papain, can be readily accumulated and trapped at temperatures below -50 degrees C. At these low temperatures, under conditions of excess substrate, the amount of p-nitrophenol liberated in the acylation reaction is equivalent to the active-site normality of the enzyme, indicating a 1:1 stoichiometry in formation of the acyl enzyme. The effect of dimethyl sulfoxide up to 7.65 M, on the intrinsic ultraviolet, fluorescence, and circular dichroic properties of the enzyme shows no evidence of any solvent-induced structural changes. All experimental observations are consistent with the conclusion that 7.65 M dimethyl sulfoxide and subzero temperatures have no deleterious effects on papain-catalyzed reactions. A related series of experiments indicate that aqueous ethanol cryosolvents up to 13.7 M (80% v/v) are also suitable. Preliminary experiments at subzero temperatures using Na-carbobenzoxy-L-lysine methyl ester suggest the existence of three enzyme-substrate intermediates which can be detected and accumulated.     

Structure/activity relationship of adenine-modified NAD derivatives with respect to porcine heart lactate dehydrogenase isozyme H4 simulated with molecular mechanics

Using a significantly simplified modification procedure, four charged analogues of the coenzyme NAD, N(1)- and N6-(2-hydroxy-3-trimethylammoniumpropyl)-NAD, N(1)- and N6-(3-sulfopropyl)-NAD were prepared. The kinetic parameters of these derivatives and N(1)-(2-aminoethyl)-NAD, N6-(2-aminoethyl)-NAD and tricyclic 1,N6-ethanoadenine-NAD, all with alterations to the adenine moiety, were determined for porcine heart lactate dehydrogenase isoenzyme H4. The coenzyme activity depends on both position and charge of the introduced groups. Modification of the N6-position leads to a 25-250-fold increase of the kcat/Km value compared to the related N(1) derivative. The kcat/Km value for 1,N6-ethanoadenine-NAD is in the range between that of N(1)-(2-aminoethyl)-NAD and N6-(2-aminoethyl)-NAD. In the case of both N(1) and N6 functionalization, the Km values increase from (3-sulfopropyl)-NAD, with a negatively charged substituent at the adenine, over (2-amino-ethyl)-NAD to (2-hydroxy-3-trimethylammoniumpropyl)-NAD with an uncharged and positively charged substituent, respectively, at the adenine. All N6 derivatives are analogues like NAD with respect to Km and/or Vmax and kcat/Km. The conformation of NAD and its derivatives was calculated and their interaction in the active site of lactate dehydrogenase was simulated using the molecular mechanics program AMBER. The significant differences in activity in correlation to porcine heart lactate dehydrogenase isoenzyme H4 could be rationalized by modelling the three-dimensional structure of the NAD site.     

Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases

The electronic absorption spectrum, susceptibility to fluoride inhibition, redox potential, and substrate turnover of several fungal laccases have been explored as a function of pH. The laccases showed a single spectrally detectable acid-base transition at pH 6-9 and a fluoride inhibition that diminished by increased pH (indicating a competition with hydroxide inhibition). Relatively small changes in the redox potentials (< or = 0.1 V) of laccase were observed over the pH 2.7-11. Under the catalysis of laccase, the apparent oxidation rates (kcat and kcat/Km) of two nonphenolic substrates, potassium ferrocyanide and 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid), decreased monotonically as the pH increased. In contrast, the apparent oxidation rates (kcat and kcat/Km) of three 2,6-dimethoxyphenols (whose pKa values range from 7.0 to 8.7) exhibited bell-shaped pH profiles whose maxima were distinct for each laccase but independent of the substrate. By correlating these pH dependences, it is proposed that the balance of two opposing effects, one generated by the redox potential difference between a reducing substrate and the type 1 copper of laccase (which correlates to the electron transfer rate and is favored for a phenolic substrate by higher pH) and another generated by the binding of a hydroxide anion to the type 2/type 3 coppers of laccase (which inhibits the activity at higher pH), contributes to the pH activity profile of the fungal laccases.     

Kinetic and mechanistic characterization of a mammalian protein-tyrosine phosphatase, PTP1

The kinetic mechanism of the hydrolysis of phosphate monoesters catalyzed by a soluble form of rat protein-tyrosine phosphatase (PTPase), PTP1, was probed with a variety of steady-state and pre-steady-state kinetic techniques. Product inhibition and 18O exchange experiments are consistent with the enzymatic reaction proceeding through two chemical steps, i.e. formation and breakdown of a covalent phosphoenzyme intermediate. The variation of kcat/Km with pH indicates that three ionizable groups are involved in enzyme substrate binding and catalysis. The first group must be deprotonated and is attributed to the second ionization of the substrate. The other two groups with pK alpha values of 5.1 and 5.5 correspond to two enzyme active site residues. The kcat-pH profiles for both p-nitrophenyl phosphate and beta-naphthyl phosphate are bell-shaped and are superimposable, with the apparent pK alpha values derived from the acidic limb and the basic limb of the profile being 4.4 and 6.8, respectively. This suggests that the rate-limiting step corresponds to the decomposition of the phosphoenzyme intermediate at all pH values. Results from leaving group dependence of kcat at two different pH values support the above conclusion. Furthermore, burst kinetics have been demonstrated with PTP1 using p-nitrophenyl phosphate as a substrate. The rate constants for the formation and the breakdown of the intermediate are 241 and 12 s-1, respectively, at pH 6.0 and 3.5 degrees C. A normal D2O solvent isotope effect (kcatH/kcatD = 1.5) is associated with the breakdown of the phosphoenzyme intermediate, indicating a solvent-derived proton in the transition state. The leaving group dependence of kcat/Km suggests that there is a strong electrophilic interaction between the enzyme and the leaving group oxygen in the transition state of the phosphorylation event. These results are compared with those of the Yersinia PTPase and suggest that the mechanism for PTPase-catalyzed phosphate monoester hydrolysis is conserved from bacterial to mammals.     

Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets

Group II intron ai5 gamma was reconstructed into a multiple-turnover ribozyme that efficiently cleaves small oligonucleotide substrates in-trans. This construct makes it possible to investigate sequence specificity, since second-order rate constants (kcat/K(m), or the specificity constant) can be obtained and compared with values for mutant substrates and with other ribozymes. The ribozyme used in this study consists of intron domains 1 and 3 connected in-cis, together with domain 5 as a separate catalytic cofactor. This ribozyme has mechanistic features similar to the first step of reverse-splicing, in which a lariat intron attacks exogenous RNA and DNA substrates, and it therefore serves as a model for the sequence specificity of group II intron mobility. To quantitatively evaluate the sequence specificity of this ribozyme, the WT kcat/Km value was compared to individual kcat/Km values for a series of mutant substrates and ribozymes containing single base changes, which were designed to create mismatches at varying positions along the two ribozyme-substrate recognition helices. These mismatches had remarkably large effects on the discrimination index (1/relative kcat/K(m)), resulting in values > 10,000 in several cases. The delta delta G++ for mismatches ranged from 2 to 6 kcal/mol depending on the mismatch and its position. The high specificity of the ribozyme is attributable to effects on duplex stabilization (1-3 kcal/mol) and unexpectedly large effects on the chemical step of reaction (0.5-2.5 kcal/mol). In addition, substrate association is accompanied by an energetic penalty that lowers the overall binding energy between ribozyme and substrate, thereby causing the off-rate to be faster than the rate of catalysis and resulting in high specificity for the cleavage of long target sequences (> or = 13 nucleotides).