ES complexes of Aeromonas aminopeptidase: direct observation by stopped-flow fluorescence

Intermediates of Aeromonas aminopeptidase are monitored through fluorescence generated by radiationless energy transfer (RET) between enzyme tryptophans and the dansyl group of the bound substrate. Upon binding of the substrate enzyme tryptophan fluorescence is quenched and substrate dansyl fluorescence enhanced. These processes are reversed upon hydrolysis of the Leu-Ala bond and release of Ala-DED from the enzyme. Stopped-flow RET kinetic analysis yields values of kcat = 36 sec-1 and Km = 3.7 microM at pH 7.5 and 20 degrees C. These values represent the highest kcat/Km ratio, 1 X 10(7) M-1 sec-1, of any substrate for Aeromonas aminopeptidase. The excellent binding properties of the peptide permit direct visualization of ES complexes even at enzyme concentrations of 10(-7) M.     

Interaction of carboxypeptidase A with carbamate and carbonate esters

The carbamate ester N-(phenoxycarbonyl)-L-phenylalanine binds well to carboxypeptidase A in the manner of peptide substrates. The ester exhibits linear competitive inhibition toward carboxypeptidase A catalyzed hydrolysis of the amide hippuryl-L-phenylalanine (Ki = 1.0 X 10(-3) M at pH 7.5) and linear noncompetitive inhibition toward hydrolysis of the specific ester substrate O-hippuryl-L-beta-phenyllactate (Ki = 1.4 X 10(-3) M at pH 7.5). Linear inhibition shows that only one molecule of inhibitor is bound per active site at pH 7.5. The hydrolysis of the carbamate ester is not affected by the presence of 10(-8)-10(-9) M enzyme (the concentrations employed in inhibition experiments), but at an enzyme concentration of 3 X 10(-6) M catalysis can be detected. The value of kcat at 30 degrees C, mu = 0.5 M, and pH 7.45 is 0.25 s-1, and Km is 1.5 X 10(-3) M. The near identity of Km and Ki shows that Km is a dissociation constant. Substrate inhibition can be detected at pH less than 7 but not at pH values above 7, which suggests that a conformational change is occurring near that pH. The analogous carbonate ester O-(phenoxycarbonyl)-L-beta-phenyllactic acid is also a substrate for the enzyme. The Km is pH independent from pH 6.5 to 9 and has the value of 7.6 X 10(-5) M in that pH region. The rate constant kcat is pH independent from pH 8 to 10 at 30 degrees C (mu = 0.5 M) with a limiting value of 1.60 s-1. Modification of the carboxyl group of glutamic acid-270 to the methoxyamide strongly inhibits the hydrolysis of O-(phenoxycarbonyl)-L-beta-phenyllactic acid. Binding of beta-phenyllactate esters and phenylalanine amides must occur in different subsites, but the ratios of kcat and kcat/Km for the structural change from hippuryl to phenoxy in each series are closely similar, which suggests that the rate-determining steps are mechanistically similar.     

Human cathepsin H: deletion of the mini-chain switches substrate specificity from aminopeptidase to endopeptidase

The mini-chain of human cathepsin H has been identified as the major structural element determining the protease's substrate specificity. A genetically engineered mutant of human cathepsin H lacking the mini-chain, des[Glu(-18)-Thr(-11)]-cathepsin H, exhibits endopeptidase activity towards the synthetic substrate Z-Phe-Arg-NH-Mec (kcat = 0.4 s(-1), Km = 92 microM, kcat/Km = 4348 M(-1) s(-1)) which is not cleaved by r-wt cathepsin H. However, the mutant enzyme shows only minimal aminopeptidase activity for H-Arg-NH-Mec (kcat = 0.8 s(-1), Km = 3.6 mM, kcat/Km = 222 M(-1) s(-1)) which is one of the best known substrates for native human cathepsin H (kcat = 2.5 s(-1), Km = 150 microM, kcat/Km = 16666 M(-1) s(-1)). Inhibition studies with chicken egg white cystatin and E-64 suggest that the mini-chain normally restricts access of inhibitors to the active site. The kinetic data on substrates hydrolysis and enzyme inhibition point out the role of the mini-chain as a structural framework for transition state stabilization of free alpha-amino groups of substrates and as a structural barrier for endopeptidase-like substrate cleavage.     

Conformation aspects of peptide interaction with proteolytic enzymes. Alpha-chymotrypsin catalyzed hydrolysis of the cyclopeptides containing leucyltyrosyl fragments]

The studies were made on the interaction of alpha-chymotrypsin with a series of cyclopeptides cyclo(-L-leucyl-L-tyrosyl-glycyln-), n=4, 6 and 8 (I, II and III respectively), and cyclo(-L-leucyl-L-tryosyl-beta-aminovalero-yl2-) (IV). Compounds I and IV are resistant to enzyme action whereas cyclopeptides II and III proved to be the substrates, their kinetic constants being Km=15.4 and 13.2 mM and kcat=0.54 and 9.53 sec-1 respectively. The binding capacity of cyclopeptides I-IV is evaluated by their competitive inhibition of alpha-chymotrypsin catalyzed hydrolysis of N-acetyl-L-tyrosine methyl ester.     

Aspartic acid 405 contributes to the substrate specificity of aminopeptidase B

Aminopeptidase B (EC 3.4.11.6, ApB) specifically cleaves in vitro the N-terminal Arg or Lys residue from peptides and synthetic derivatives. Ap B was shown to have a consensus sequence found in the metallopeptidase family. We determined the putative zinc binding residues (His324, His328, and Glu347) and the essential Glu325 residue for the enzyme using site-directed mutagenesis (Fukasawa, K. M., et al. (1999) Biochem. J. 339, 497-502). To identify the residues binding to the amino-terminal basic amino acid of the substrate, rat cDNA encoding ApB was cloned into pGEX-4T-3 so that recombinant protein was expressed as a GST fusion protein. Twelve acidic amino acid residues (Glu or Asp) in ApB were replaced with a Gln or Asn using site-directed mutagenesis. These mutants were isolated to characterize the kinetic parameters of enzyme activity toward Arg-NA and compare them to those of the wild-type ApB. The catalytic efficiency (kcat/Km) of the mutant D405N was 1.7 x 10(4) M(-1) s(-1), markedly decreased compared with that of the wild-type ApB (6.2 x 10(5) M(-1) s(-1)). The replacement of Asp405 with an Asn residue resulted in the change of substrate specificity such that the specific activity of the mutant D405N toward Lys-NA was twice that toward Arg-NA (in the case of wild-type ApB; 0.4). Moreover, when Asp405 was replaced with an Ala residue, the kcat/Km ratio was 1000-fold lower than that of the wild-type ApB for hydrolysis of Arg-NA; in contrast, in the hydrolysis of Tyr-NA, the kcat/Km ratios of the wild-type (1.1 x 10(4) M(-1) s(-1)) and the mutated (8.2 x 10(3) M(-1) s(-1)) enzymes were similar. Furthermore, the replacement of Asp-405 with a Glu residue led to the reduction of the kcat/Km ratio for the hydrolysis of Arg-NA by a factor of 6 and an increase of that for the hydrolysis of Lys-NA. Then the kcat/Km ratio of the D405E mutant for the hydrolysis of Lys-NA was higher than that for the hydrolysis of Arg-NA as opposed to that of wild-type ApB. These data strongly suggest that the Asp 405 residue is involved in substrate binding via an interaction with the P1 amino group of the substrate's side chain.     

Activation of human Hageman factor by Pseudomonas aeruginosa elastase in the presence or absence of negatively charged substance in vitro.

Human Hageman factor, a plasma proteinase zymogen, was activated in vitro under a near physiological condition (pH 7.8, ionic strength I = 0.14, 37 degrees C) by Pseudomonas aeruginosa elastase, which is a zinc-dependent tissue destructive neutral proteinase. This activation was completely inhibited by a specific inhibitor of the elastase, HONHCOCH(CH2C6H5)CO-Ala-Gly-NH2, at a concentration as low as 10 microM. In this activation Hagemen factor was cleaved, in a limited fashion, liberating two fragments with apparent molecular masses of 40 and 30 kDa, respectively. The appearance of the latter seemed to correspond chronologically to the generation of activated Hageman factor. Kinetic parameters of the enzymatic activation were kcat = 5.8 x 10(-3) s-1, Km = 4.3 x 10(-7) M and kcat/Km = 1.4 x 10(4) M-1 x s-1. This Km value is close to the plasma concentration of Hageman factor. Another zinc-dependent proteinase, P. aeruginosa alkaline proteinase, showed a negligible Hageman factor activation. In the presence of a negatively charged soluble substance, dextran sulfate (0.3-3 micrograms/ml), the activation rate by the elastase increased several fold, with the kinetic parameters of kcat = 13.9 x 10(-3) s-1, Km = 1.6 x 10(-7) M and kcat/Km = 8.5 x 10(4) M-1 x s-1. These results suggested a participation of the Hageman factor-dependent system in the inflammatory response to pseudomonal infections, due to the initiation of the system by the bacterial elastase.     

Mechanistic requirements for the efficient enzyme-catalyzed hydrolysis of thiosialosides

The sialidase from Micromonospora viridifaciens has been found to catalyze the hydrolysis of aryl 2-thio-alpha-D-sialosides with remarkable efficiency: the first- and second-order rate constants, kcat and kcat/Km, for the enzyme-catalyzed hydrolysis of PNP-S-NeuAc are 196 +/- 5 s(-1) and (6.7 +/- 0.7) x 10(5) M(-1) s(-1), respectively. A reagent panel of eight aryl 2-thio-alpha-D-sialosides was synthesized and used to probe the mechanism for the M. viridifaciens sialidase-catalyzed hydrolysis reaction. In the case of the wild-type enzyme, the derived Br?nsted parameters (beta(lg)) on kcat and kcat/Km are -0.83 +/- 0.11 and -1.27 +/- 0.17 for substrates with thiophenoxide leaving groups of pKa values > or = 4.5. For the general-acid mutant, D92G, the derived beta(lg) value on kcat for the same set of leaving groups is -0.82 +/- 0.12. When the conjugate acid of the departing thiophenol was < or = 4.5, the derived Br?nsted slopes for both the wild-type and the D92G mutant sialidase were close to zero. In contrast, the nucleophilic mutant, Y370G, did not display a similar break in the Br?nsted plots, and the corresponding values for beta(lg), for the three most reactive aryl 2-thiosialosides, on kcat and kcat/Km are -0.76 +/- 0.28 and -0.84 +/- 0.04, respectively. Thus, for the Y370G enzyme glycosidic C-S bond cleavage is rate-determining for both kcat and kcat/Km, whereas, for both the wild-type and D92G mutant enzymes, the presented data are consistent with a change in rate-determining step from glycosidic C-S bond cleavage for substrates in which the pKa of the conjugate acid of the leaving group is > or = 4.5, to either deglycosylation (kcat) or a conformational change that occurs prior to C-S bond cleavage (kcat/Km) for the most activated leaving groups. Thus, the enzyme-catalyzed hydrolysis of 2-thiosialosides is strongly catalyzed by the nucleophilic tyrosine residue, yet the C-S bond cleavage does not require the conserved aspartic acid residue (D92) to act as a general-acid catalyst.     

Dependence of the P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry. Quantification of selectivity in the catalysed hydrolysis of the enantiomeric N-acetylphenylalanylglycine 4-nitroanilides.

1. N-Acetyl-L-phenylalanylglycine 4-nitroanilide and its D-enantiomer were synthesized and characterized and used as substrates with which to evaluate stereochemical selectivity in papain (EC 3.4.22.2)-catalysed hydrolysis. 2. Kinetic analysis at pH 6.0, I 0.1, 8.3% (v/v) NN-dimethylformamide and 25 degrees C by using initial-rate data with [S] much less than Km and weighted non-linear regression provided values of kcat./Km for the catalysed hydrolysis of both enantiomers as (kcat./Km)L = 2040 +/- 48 M-1.S-1 and (kcat./Km)D = 5.9 +/- 0.07 M-1.S-1. These data, taken together with individual values of kcat. and Km for the hydrolysis of the L-enantiomer (a) estimated in the present work as kcat. = 3.2 +/- 1.2 S-1 and Km = 1.5 +/- 0.6 mM and (b) reported by Lowe & Yuthavong [(1971) Biochem. J. 124, 107-115] for the reaction at pH 6.0 in 10% (v/v) NN-dimethylformamide and 35 degrees C, as kcat. = 1.3 +/- 0.2 S-1 and Km = 0.88 +/- 0.1 mM, suggest that (kcat./Km)L congruent to 2000 M-1.S-1 and thus that (kcat./Km)L/(kcat./Km)D congruent to 330.3. Model building indicates that both enantiomeric 4-nitroanilides can bind to papain such that the phenyl ring of the N-acetylphenylalanyl group makes hydrophobic contacts in the S2 subsite with preservation of mechanistically relevant hydrogen-bonding interactions and that the main difference is in the positioning of the beta-methylene group. 4. The dependence of P2-S2 stereochemical selectivity of papain on the nature of the catalytic-site chemistry for reactions involving derivatives of N-acetylphenylalanine is discussed. The variation in the index of stereochemical selectivity (ratio of the appropriate kinetic or thermodynamic parameter for a given pair of enantiomeric ligands), from 330 for the overall acylation process of the catalytic act, through 40 and 31 for the reaction at electrophilic sulphur in 2-pyridyl disulphides respectively without and with assistance by (His-159)-Im(+)-H, to 5 for the formation of thiohemiacetal adducts by reaction at aldehydic carbon, is interpreted in terms of the extent to which conformational variation of the bound ligand in the catalytic-site region permits the binding mode of the -CH2-Ph group of the D-enantiomer to approach that of the L-enantiomer.     

Catalytic properties of human Lys77-plasmin. A comparative steady-state and pre-steady-state study

Values of kinetic parameters for the hydrolysis of esters and p-nitroanilides of L-lysine and L-arginine catalyzed by the Lys77 form of human plasmin (EC 3.4.21.7) have been determined between pH 5.5 and 8 (I = 0.1 M) at 21 +/- 0.5 degrees C. Over the whole pH range explored, Lys77-plasmin catalysis conforms to simple Michaelis-Menten kinetics, and steady-state and pre-steady-state data may be consistently fitted to the minimum three-step mechanism: E + S in equilibrium (k+1/k-1)E X S----(k+2)E X P + P1----(k+3)E + P2 In spite of the higher specificity of lysyl derivatives for Lys77-plasmin rather than the arginyl ones, kinetic parameters also depend on the nature of the N-alpha substituent and/or of the alcoholic or p-nitroanilidic moiety of the substrate. Among the esters and anilides considered, ZLysONp shows the most favourable kinetic parameters and may be the substrate of choice of Lys77-plasmin, in that it allows the determination of the enzyme concentration as low as 2 X 10(-9) M (about 1 X 10(-3) CU/ml), at the optimum pH value (approx. 8). Between pH 5.5 and 8, the pH profiles of kcat and kcat/Km for the Lys77-plasmin-catalyzed hydrolysis of ZLysONp and ZArgONp reflect the ionization of a single group (probably His-602 involved in the active site) with pKa values ranging between 6.4 and 6.6; at variance, values of Km are pH-independent.     

Purification and partial characterization of an alpha-chymotrypsin-like protease of rat peritoneal mast cells.

An alpha-chymotrypsin-like enzyme was isolated from mast cells of the rat peritoneal cavity by extraction with 0.8 M potassium phosphate, 2 per cent protamine sulfate followed by affinity chromatography on hen ovoinhibitor-agarose and adsorption on barium sulfate. This procedure yielded over 9 mg of protease from the peritoneal lavage fluid of 100 rats, equivalent to 44 per cent of the initial activity. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, analytical isoelectric focusing, and amino-terminal sequence analysis. The protease contains no covalently bound carbohydrate and has a molecular weight of approximately 26,000. The enzyme molecule is a single polypeptide chain with an amino-terminal sequence homologous to that of the B chain of bovine alpha-chymotrypsin. The kinetic parameters, Km and kcat, for the hydrolysis of N-benzoyl-L-tyrosine ethyl ester were determined at pH 8.0 and 25 degrees C as 1.1 X 10(-3) M and 84 sec-1, respectively. The value of the second-order rate constant for inactivation of mast cell protease by diisopropylphosphofluoridate was 300 times lower than for bovine alpha-chymotrypsin.     

Use of a fluorescence plate reader for measuring kinetic parameters with inner filter effect correction

A general method is presented here for the determination of the Km, kcat, and kcat/Km of fluorescence resonance energy transfer (FRET) substrates using a fluorescence plate reader. A simple empirical method for correcting for the inner filter effect is shown to enable accurate and undistorted measurements of these very important kinetic parameters. Inner filter effect corrected rates of hydrolysis of a FRET peptide substrate by hepatitis C virus (HCV) NS3 protease at various substrate concentrations enabled measurement of a Km value of 4.4 +/- 0.3 microM and kcat/Km value of 96,500 +/- 5800 M-1 s-1. These values are very close to the HPLC-determined Km value of 4.6 +/- 0.7 microM and kcat/Km value of 92,600 +/- 14,000 M-1 s-1. We demonstrate that the inner filter effect correction of microtiter plate reader velocities enables rapid measurement of Ki and Ki' values and kinetic inhibition mechanisms for HCV NS3 protease inhibitors.     

Diffusion-dependent rates for the hydrolysis reaction catalyzed by glyoxalase II from rat erythrocytes

Glyoxalase II from rat erythrocytes is a near optimal catalyst for the hydrolysis of S-D-lactoylglutathione in the sense that the magnitude of kcat/Km is limited, in large part, by the rate constant for diffusion-controlled encounter between substrate and active site. The experimental basis for this conclusion is derived from the dependencies of the kinetic properties of the enzyme on solution viscosity (pH 7, Ic = 0.1 M, 25 degrees C). When sucrose is used as a viscogenic agent, kcat/Km for S-D-lactoylglutathione (8.8 x 10(5) M-1 s-1) decreases markedly with increasing solution viscosity. This effect appears not to be due to a sucrose-induced change in the intrinsic kinetic properties of the enzyme, since kcat/Km for the slow substrate S-acetylglutathione (3.7 x 10(4) M-1 s-1) is nearly independent of solution viscosity. Quantitative treatment of the data using Stoke's law indicates that the rate of hydrolysis of S-D-lactoylglutathione will be approximately 50% diffusion limited when [substrate] much less than Km; the encounter complex between enzyme and substrate partitions nearly equally between product formation and dissociation to form free enzyme and substrate. The same conclusion is reached when glycerol is used as a viscogenic agent, once the apparent activation effect of glycerol on the intrinsic activity of the enzyme is taken into account. Finally, the rate of formation of the encounter complex between substrate and active site may be governed to a significant extent by charge-charge interactions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic properties of triose-phosphate isomerase from Trypanosoma brucei brucei. A comparison with the rabbit muscle and yeast enzymes

The kinetic properties of Trypanosoma brucei brucei triose-phosphate isomerase are compared with those of the commercially available rabbit muscle and yeast enzymes and with published data on the chicken muscle enzyme. With glyceraldehyde 3-phosphate as substrate Km = 0.25 +/- 0.05 mM and kcat = 3.7 X 10(5) min-1. With dihydroxyacetone phosphate as substrate Km = 1.2 +/- 0.1 mM and kcat = 6.5 X 10(4) min-1. The pH dependence of Km and Vmax at 0.1 M ionic strength is in agreement with the results published for the yeast and chicken muscle enzymes. At ionic strength below 0.05 M the effect of a charged group specific for the trypanosomal enzyme and absent from the yeast and rabbit muscle enzymes becomes detectable. This effect significantly increases Km whereas Vmax becomes slightly higher. Trypanosomal triose-phosphate isomerase is inhibited by sulphate, phosphate and arsenate ions, by 2-phosphoglycolate and a number of documented inhibitors in the same concentration range as are the other triose-phosphate isomerases. The trypanocidal drug, Suramin inhibits T. brucei and rabbit muscle triose-phosphate isomerase to the same extent while leaving the yeast enzyme relatively unaffected.     

Human cathepsin F: expression in baculovirus system, characterization and inhibition by protein inhibitors

Recombinant full-length human procathepsin F, produced in the baculovirus expression system, was partially processed during the purification procedure to a form lacking the N-terminal cystatin-like domain and activated with pepsin. Active cathepsin F efficiently hydrolyzed Z-FR-MCA (kcat/Km=106 mM(-1) s(-1)) and Bz-FVR-MCA (kcat/Km=8 mM(-1) s(-1)), whereas hydrolysis of Z-RR-MCA was very slow (kcat/Km<0.2 mM(-1) s(-1)). Cathepsin F was rapidly and tightly inhibited by cystatin C, chicken cystatin and equistatin with Ki values in the subnanomolar range (0.03-0.47 nM), whereas L-kininogen was a less strong inhibitor of the enzyme (Ki=4.7 nM). Stefin A inhibited cathepsin F slowly (kass=1.6 x 10(5) M(-1) s(-1)) and with a lower affinity (Ki=25 nM). These data suggest that cathepsin F differs from other related endopeptidases by considerably weaker inhibition by stefins.     

Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein

We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.     

Partial characterization of 2 extracellular aminopeptidases (arylamidases) from the dermatophyte Keratinomyces ajelloi]

Two aminopeptidases AP1 and AP2 have been isolated from Keratinomyces ajelloi filtrates. The molecular weight is about 27 000 for AP1 and 23 000 for AP2. Both aminopeptidases present maximum activity at pH 9.35 but 50 p. 100 of maximum activity is observed between pH 7.5 and pH 8.5. Km values measured at pH 9.35 with L-leucine-p-nitroanilide as substrate are 0.38 X 10(-3) M for AP1 and 0.43 X 10(-3) M for AP2. kcat at the same pH are 63.6 sec.-1 for AP-1 and 62.8 sec-1 for AP2. Both aminopeptidases are inhibited by mercuric chloride, o-phenanthroline, dithiothreitol and 2-mercaptoethanol. Some of their characters make them similar to Streptomyces griseus pronase aminopeptidases.     

New substrates for enteropeptidase. I. Biologically active hepta-nonapeptides

Enteropeptidase (enterokinase, EC 3.4.21.9) hydrolyzes peptide bonds formed by carboxyl groups of Lys or Arg residue provided that less than four negatively charged amino acid residues are in positions P2-P5 of its substrate. We determined the kinetic parameters of three substrates of this type: human angiotensin II (AT) (DR decreases VYIHPF) and the Hb(2-8) (LTAEEK decreases A) and Hb(1-9) (MLTAEEK decreases AA) peptides of the cattle hemoglobin beta-chain. The Km values for all the substrates (approximately 10(-3) M) were one order of magnitude higher than those of the typical synthetic substrates of enteropeptidase or chimeric proteins with the -DDDDK- full-size linker (Km approximately 10(-4) M). The kcat values for AT and Hb(2-8) were also close and low (approximately 30 min-1). The general hydrolysis efficiency of such substrates is no more than 1% of the corresponding value for the typical peptide and protein substrates of the enteropeptidase. However, the elongation of Hb(2-8) peptide by one amino acid residue from its N- or C-terminus results in a dramatic increase in the catalytic efficiency of the hydrolysis: the kcat value for Hb(1-9) is 1510 min-1, which means that it is hydrolyzed only three times less effective than the chimeric protein with the full-size linker.     

Transient state kinetic studies of the MutT-catalyzed nucleoside triphosphate pyrophosphohydrolase reaction

The MutT pyrophosphohydrolase, in the presence of Mg2+, catalyzes the hydrolysis of nucleoside triphosphates by nucleophilic substitution at Pbeta, to yield the nucleotide and PP(i). The best substrate for MutT is the mutagenic 8-oxo-dGTP, on the basis of its Km being 540-fold lower than that of dGTP. Product inhibition studies have led to a proposed uni-bi-iso kinetic mechanism, in which PP(i) dissociates first from the enzyme-product complex (k3), followed by NMP (k4), leaving a product-binding form of the enzyme (F) which converts to the substrate-binding form (E) in a partially rate-limiting step (k5) [Saraswat, V., et al. (2002) Biochemistry 41, 15566-15577]. Single- and multiple-turnover kinetic studies of the hydrolysis of dGTP and 8-oxo-dGTP and global fitting of the data to this mechanism have yielded all of the nine rate constants. Consistent with an "iso" mechanism, single-turnover studies with dGTP and 8-oxo-dGTP hydrolysis showed slow apparent second-order rate constants for substrate binding similar to their kcat/Km values, but well below the diffusion limit (approximately 10(9) M(-1) s(-1)): k(on)app = 7.2 x 10(4) M(-1) s(-1) for dGTP and k(on)app = 2.8 x 10(7) M(-1) s(-1) for 8-oxo-dGTP. These low k(on)app values are fitted by assuming a slow iso step (k5 = 12.1 s(-1)) followed by fast rate constants for substrate binding: k1 = 1.9 x 10(6) M(-1) s(-1) for dGTP and k1 = 0.75 x 10(9) M(-1) s(-1) for 8-oxo-dGTP (the latter near the diffusion limit). With dGTP as the substrate, replacing Mg2+ with Mn2+ does not change k1, consistent with the formation of a second-sphere MutT-M2+-(H2O)-dGTP complex, but slows the iso step (k5) 5.8-fold, and its reverse (k(-5)) 25-fold, suggesting that the iso step involves a change in metal coordination, likely the dissociation of Glu-53 from the enzyme-bound metal so that it can function as the general base. Multiple-turnover studies with dGTP and 8-oxo-dGTP show bursts of product formation, indicating partially rate-limiting steps following the chemical step (k2). With dGTP, the slow steps are the chemical step (k2 = 10.7 s(-1)) and the iso step (k5 = 12.1 s(-1)). With 8-oxo-dGTP, the slow steps are the release of the 8-oxo-dGMP product (k4 = 3.9 s(-1)) and the iso step (k5 = 12.1 s(-1)), while the chemical step is fast (k2 = 32.3 s(-1)). The transient kinetic studies are generally consistent with the steady state kcat and Km values. Comparison of rate constants and free energy diagrams indicate that 8-oxo-dGTP, at low concentrations, is a better substrate than dGTP because it binds to MutT 395-fold faster, dissociates 46-fold slower, and has a 3.0-fold faster chemical step. The true dissociation constants (KD) of the substrates from the E-form of MutT, which can now be obtained from k(-1)/k1, are 3.5 nM for 8-oxo-dGTP and 62 microM for dGTP, indicating that 8-oxo-dGTP binds 1.8 x 10(4)-fold tighter than dGTP, corresponding to a 5.8 kcal/mol lower free energy of binding.     

Kinetic study of the action of snake venom phospholipase A2 on human serum high density lipoprotein 3.

The hydrolysis of the phospholipids of intact human serum high density lipoprotein 3 (HDL3) by pure alpha-phospholipase A2 from Crotalus adamanteus was studied by pH-stat titration. The enzyme quantitatively hydrolyzed phosphatidylcholine and phosphatidylethanolamine and left sphinogomyelin intact, yielding a stable and water-soluble modified HDL. Lysophospholipids and free fatty acids, the products of hydrolysis, remained in the lipoprotein. When 1 mol of defatted bovine serum albumin/mol of substrate phospholipids was added to the reaction mixture, up to 60% of the fatty acids and 85% of the lysophospholipids were removed from the modified lipoprotein. The immunological reactivity of the hydrolyzed HDL remained unaltered in both the presence and absence of albumin. The changes in the physical properties of the lipoprotein during hydrolysis were rather small, the most notable being an increase in the hydrated density and in the electrophoretic mobility in alkaline buffers. The hydrolysis followed an apparent first order time course with product inhibition (KI) and yielded values of kcat/Km = 7 X 10(5 M(-1)s(-1) and KI congruent to 1 X 10(-4) M. Addition of albumin to the reaction mixture relieved the product inhibition without any alteration of the kinetic parameters. High concentrations of albumin protected some of the substrate phospholipids from hydrolysis, presumably through complexation to the lipoprotein. The Arrhenius plot for the experimental first order rate constant in the absence of albumin (kexp = kcat (KI/Km)) was linear between 15 degrees and 47 degrees, indicating the absence of any phospholipid phase transitions and yielding an activation energy of 15.2 kcal/mol. From the accessibility of the HDL phospholipids to phospholipase A2 one concludes that the phosphatidylcholine and phosphatidylethanolamine are located at, or are in rapid equilibrium with, the surface of this lipoprotein. It also appears that these phospholipids are not essential for maintaining the supramolecular properties of the lipoprotein in vitro. Thsu the study of the modified Hdl should provide valuable information concenring the structure and function of this lipoprotein particularly with regard to the role played by shiingomyelin.     

Kinetics of L-gamma-Glu-pNA hydrolysis by destabilase, the enzyme from the medicinal leech Hirudo medicinalis

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).