Analysis of the ACP1 gene product: classification as an FMN phosphatase.

The relationship between the ACP1 gene product, an 18kDa acid phosphatase (E.C. 3.1.3.2) postulated to function as a protein tyrosyl phosphatase, and the cellular flavin mononucleotide (FMN) phosphatase has been examined in vitro and by using cultured Chinese hamster ovary (CHO) cells. Kinetic analysis indicated that at pH 6 the acid phosphatase utilized a variety of phosphate monoesters as substrates. While small molecules such as FMN were effectively utilized as substrates (kcat/Km = 7.3 x 10(3) s-1M-1), the tyrosyl phosphorylated form of the adipocyte lipid binding protein was a relatively poor substrate (kcat/Km = 1.7 x 10(-1) s-1M-1) suggesting a role for the phosphatase in flavin metabolism. Fractionation of CHO cell extracts revealed that 90% of the FMN phosphatase activity was soluble and that all of the soluble activity eluted from a Sephadex G-75 column with the acid phosphatase. All of the soluble FMN phosphatase activity was inhibited by immunospecific antibodies directed against the bovine heart ACP1 gene product. These results suggest that the ACP1 gene product functions cellularly not as a protein tyrosyl phosphatase but as a soluble FMN phosphatase.     

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.     

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart.

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37 degrees C. With the exception of alpha-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km) vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km) = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

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)     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Leaving group dependence in the phosphorylation of Escherichia coli alkaline phosphatase by monophosphate esters

Values of kcat and Km have been measured for the Escherichia coli alkaline phosphatase catalyzed hydrolysis of 18 aryl and 12 alkyl monophosphate esters at pH 8.00 and 25 degrees C. A Br?nsted plot of log (kcat/Km) (M-1 s-1) vs. the pK of the leaving hydroxyl group exhibits two regression lines: log (kcat/Km) = -0.19 (+/- 0.02) pKArOH + 8.14 (+/- 0.15) log (kcat/Km) = -0.19 (+/- 0.01) pKROH + 5.89 (+/- 0.17) Alkyl phosphates with aryl or large lipophilic side chains are not correlated by the above equations and occupy positions intermediate between the two lines. The observed change in effective charge on the leaving oxygen of the ester (-0.2) is very small, consistent with substantial electrophilic participation of the enzyme with this atom. Cyclohexylammonium ion is a noncompetitive inhibitor against 4-nitrophenyl phosphate substrate at pH 8.00, and neutral phenol is a competitive inhibitor (Ki = 82.6 mM); these data and the 100-fold larger reactivity of aryl over alkyl esters are consistent with the existence of a lipophilic binding site for the leaving group of the substrate. The absence of a major steric effect in kcat/Km for substituted aryl esters confirms that the leaving group in the enzyme--substrate complex points away from the surface of the enzyme. Arguments are advanced to exclude a dissociative mechanism (involving a metaphosphate ion) for the enzyme-catalyzed substitution at phosphorus.     

Alpha-thrombin-catalyzed hydrolysis of fibrin I. Alternative binding modes and the accessibility of the active site in fibrin I-bound alpha-thrombin

Steady-state kinetic parameters were determined for the action of human alpha-thrombin on human fibrin I polymer, an intermediate in the alpha-thrombin-catalyzed conversion of fibrinogen to the fibrin matrix of blood clots during the terminal phase of the blood clotting cascade. Values of 49 s-1 and 7.5 microM were determined (at 37 degrees C, pH 7.4, gamma/2 0.17) for kcat and Km, respectively. Studies of the effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of the fluorogenic substrate N-p-Tos-Gly-L-Pro-L-Arg-7-amido-4-methylcoumarin (tos-GPR-amc) and the effect of fibrin I on the reaction of alpha-thrombin with antithrombin III (AT) were presented which indicate that the active site of alpha-thrombin is accessible while it is bound to its substrate fibrin I. Fibrin I inhibited alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc in a manner inconsistent with the pure competitive inhibition expected for an alternative substrate, whereas fibrinogen, an alpha-thrombin substrate, behaved as a pure competitive inhibitor of the alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc. The effect of fibrin I on alpha-thrombin-catalyzed hydrolysis of tos-GPR-amc was shown to be consistent with alpha-thrombin binding to fibrin I in alternative orientations. In one orientation both the active site and a site distinct from the active site (an exosite) of alpha-thrombin are occupied by fibrin I. In the other orientation only the exosite of alpha-thrombin is occupied and the active site is freely accessible to other substrates. The values of both kcat (21 s-1) and Km (less than 0.23 microM) determined for fibrin I-bound alpha-thrombin acting on tos-GPR-amc were decreased relative to the values of kcat (180 s-1) and Km (7.3 microM) observed for the action of uncomplexed alpha-thrombin on tos-GPR-amc. This observation suggests that the active site of alpha-thrombin is altered in fibrin I-bound alpha-thrombin. Studies of the effect of fibrin I on the reaction of AT with alpha-thrombin (at 37 degrees C, pH 7.4, gamma/2 0.17) indicated that when alpha-thrombin is bound to fibrin I in an orientation where the active site of alpha-thrombin is accessible, AT reacts with alpha-thrombin with a rate constant (greater than 4.2 x 10(4) M-1 s-1) that is greater than the rate constant (1.5 x 10(4) M-1 s-1) for reaction of AT with the free enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetic parameters for the elimination reaction catalyzed by triosephosphate isomerase and an estimation of the reaction's physiological significance

Kinetic parameters for triosephosphate isomerase catalysis of the elimination reaction of an equilibrium mixture of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate (DGAP) to form methylglyoxal and phosphate ion are reported for the enzyme from rabbit muscle. Pseudo-first-order rate constants for the disappearance of substrate (kelim) were determined for reactions at [Enzyme] much greater than [Substrate]. The second-order rate constant kEnz = 10.1 M-1 s-1 was determined from a plot of kelim against enzyme concentration. The kinetic parameters, determined from a steady-state kinetic analysis at [Substrate] much greater than [Enzyme], are kcat = 0.011 s-1, Km = 0.76 mM, and kcat/Km = 14 M-1 s-1. The estimated rate-constant ratio for partitioning of the enzyme-bound intermediate between protonation at carbon 2 and elimination, 1,000,000, is much larger than the ratio of 6.5 determined for the reaction of the enediolate phosphate in a loose complex with quinuclidinonium cation, a small buffer catalyst. There is a 10(5)-10(8)-fold decrease in the rate constant for the elimination reaction of the enediolate phosphate when this species binds to triosephosphate isomerase. The kinetic parameters for the elimination reaction catalyzed by the native triosephosphate isomerase and for the reaction catalyzed by a mutant form of the enzyme, which is missing a segment that forms hydrogen bonds with the phosphate group of substrate [Pompliano, D. L., Peyman, A., & Knowles, J. R. (1990) Biochemistry 29, 3186-3194] are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and analytical use of 3-carboxypropionyl-alanyl-alanyl-valine-4-nitroanilide: a specific substrate for human leukocyte elastase

A simple synthesis is described for 3-carboxypropionyl-Ala-Ala-Val-4-nitroanilide, a convenient and very specific substrate for human leukocyte elastase (Km = 1.0mM, kcat = 8.7 s-1). The substrate does not undergo appreciable spontaneous hydrolysis. It is not cleaved by trypsin or chymotrypsin and only rather slowly by porcine pancreatic elastase (Km = 9.1mM, kcat = 1.4 s-1).     

Effects of secondary interactions on the kinetics of peptide and peptide ester hydrolysis by tissue kallikrein and trypsin

Kinetic constants for the hydrolysis by porcine tissue beta-kallikrein B and by bovine trypsin of a number of peptides related to the sequence of kininogen (also one containing a P2 glycine residue instead of phenylalanine) and of a series of corresponding arginyl peptide esters with various apolar P2 residues have been determined under strictly comparative conditions. kcat and kcat/Km values for the hydrolysis of the Arg-Ser bonds of the peptides by trypsin are conspicuously high. kcat for the best of the peptide substrates, Ac-Phe-Arg-Ser-Val-NH2, even reaches kcat for the corresponding methyl ester, indicating rate-limiting deacylation also in the hydrolysis of a peptide bond by this enzyme. kcat/Km for the hydrolysis of the peptide esters with different nonpolar L-amino acids in P2 is remarkably constant (range 1.7), as it is for the pair of the above pentapeptides with P2 glycine or phenylalanine. kcat for the ester substrates varies fivefold, however, being greatest for the P2 glycine compounds. Obviously, an increased potential of a P2 residue for interactions with the enzyme lowers the rate of deacylation. In contrast to results obtained with chymotrypsin and pancreatic elastase, trypsin is well able to tolerate a P3 proline residue. In the hydrolysis of peptide esters, tissue kallikrein is definitely superior to trypsin. Conversely, peptide bonds are hydrolyzed less efficiently by tissue kallikrein and the acylation reaction is rate-limiting. The influence of the length of peptide substrates is similar in both enzymes and indicates an extension of the substrate recognition site from subsite S3 to at least S'3 of tissue kallikrein and the importance of a hydrogen bond between the P3 carbonyl group and Gly-216 of the enzymes. Tissue kallikrein also tolerates a P3 proline residue well. In sharp contrast to the behaviour of trypsin is the very strong influence of the P2 residue in tissue-kallikrein-catalyzed reactions. kcat/Km varies 75-fold in the series of the dipeptide esters with nonpolar L-amino acid residues in P2, a P2 glycine residue furnishing the worst and phenylalanine the best substrate, whereas this exchange in the pentapeptides changes kcat/Km as much as 730-fold. This behaviour, together with the high value of kcat/Km for Ac-Phe-Arg-OMe of 3.75 X 10(7) M-1 s-1, suggests rate-limiting binding (k1) in the hydrolysis of the best ester substrates.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae.

A cytosolic aldo-keto reductase was purified from Saccharomyces cerevisiae ATCC 26602 to homogeneity by affinity chromatography, chromatofocusing, and hydroxylapatite chromatography. The relative molecular weights of the aldo-keto reductase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography were 36,800 and 35,000, respectively, indicating that the enzyme is monomeric. Amino acid composition and N-terminal sequence analysis revealed that the enzyme is closely related to the aldose reductases of xylose-fermenting yeasts and mammalian tissues. The enzyme was apparently immunologically unrelated to the aldose reductases of other xylose-fermenting yeasts. The aldo-keto reductase is NADPH specific and catalyzes the reduction of a variety of aldehydes. The best substrate for the enzyme is the aromatic aldehyde p-nitrobenzaldehyde (Km = 46 microM; kcat/Km = 52,100 s-1 M-1), whereas among the aldoses, DL-glyceraldehyde was the preferred substrate (Km = 1.44 mM; kcat/Km = 1,790 s-1 M-1). The enzyme failed to catalyze the reduction of menadione and p-benzoquinone, substrates for carbonyl reductase. The enzyme was inhibited only slightly by 2 mM sodium valproate and was activated by pyridoxal 5'-phosphate. The optimum pH of the enzyme is 5. These data indicate that the S. cerevisiae aldo-keto reductase is a monomeric NADPH-specific reductase with strong similarities to the aldose reductases.     

Purification and properties of the phosphotriesterase from Pseudomonas diminuta

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.     

The reaction of acetyldithio-CoA, a readily enolized analog of acetyl-CoA with thiolase from Zoogloea ramigera

Acetyldithio-CoA has been shown to be a competent nucleophilic substrate but not an electrophilic substrate for the Claisen condensation catalyzed by thiolase, which normally dimerizes acetyl (Ac)-CoA to acetoacetyl-CoA. Acting as the nucleophile, the kcat/Km for dithioacetyl-CoA is comparable to that of Ac-CoA, the normal substrate. With acetoacetyl-pantetheine acetylating the thiolase to provide the electrophile, the kcat and kcat/Km for the Claisen condensation are 2.1 s-1 and 8.3 X 10(4) M-1 s-1, respectively. The product of the reaction is 3-ketobutyryldithio-CoA. The 3-ketobutyryldithio-CoA has a spectrally determined pKa of 6.55 and the enolate has a lambda max of 357 nm, epsilon 357 = 21,000 cm-1 M-1. Product analysis indicates that acetyldithio-CoA does not serve as the electrophilic partner in the enzymic condensation. This failure is attributed to the inability demonstrated in this study of acetyldithio-CoA to thioacetylate the active site Cys89 of the Zoogloea ramigera thiolase. 1H NMR studies in D2O indicate that thiolase catalyzes the exchange of the alpha-hydrogens, without Cys89 being acetylated, with a rate of 0.63 +/- 0.25 s-1. In the presence of a large excess of acetoacetyl-pantetheine, present to acetylate Cys89 and prevent the thiolytic back reaction, solvent exchange of the alpha-hydrogens can still be detected by observing the isotope-shifted 13C NMR spectrum of [2-13C]acetyldithio-CoA. The exchange of the acetyldithio-CoA alpha-hydrogens with solvent promoted by the acetylated enzyme, must proceed at a rate comparable to that of the condensation reaction.     

Interaction between bovine trypsin and a synthetic peptide containing 28 residues of the bait region of human alpha 2-macroglobulin.

The time course of the interaction between trypsin and a synthetic peptide corresponding to a segment (residues 676-703) of the bait region (residues 666-706) of human alpha 2-macroglobulin (alpha 2M) was studied by measuring the generation of cleavage products as a function of time by HPLC. Three primary cleavage sites for trypsin were present in the synthetic peptide. The fastest cleavage occurred at the bond corresponding to Arg696-Leu in alpha 2M with an estimated kcat/Km = 1-2 x 10(6) M-1.s-1. This value is of the same magnitude as that characterizing the interaction of alpha 2M and trypsin when taking into account the fact that alpha 2M is a tetramer, kcat/Km = 5 x 10(6) M-1.s-1 [Christensen, U. & Sottrup-Jensen, L. (1984) Biochemistry 23, 6619-6626]. The values of kcat/Km for cleavage at bonds corresponding to Arg681-Val and Arg692-Gly in alpha 2M were 1.5 x 10(5) M-1.s-1 and 1.3 x 10(5) M-1.s-1, respectively. Cleavage of intermediate product peptides was slower, with kcat/Km in the range 13-1.3 x 10(6) M-1.s-1. The value of Km determined for fast cleavage in the synthetic peptide was 8-10 microM. 1H-NMR spectroscopy indicated no ordered structure of the peptide. Hence, the very fast cleavage of the peptide is compatible with a loose structure that readily adopts a conformation favorable for recognition and cleavage by trypsin.     

Substrate and inhibitor studies on proteinase 3.

Various amino acid and peptide thioesters were tested as substrates for human proteinase 3 and the best substrate is Boc-Ala-Ala-Nva-SBzl with a kcat/Km value of 1.0 x 10(6) M-1.s-1. Boc-Ala-Ala-AA-SBzl (AA = Val, Ala, or Met) are also good substrates with kcat/Km values of (1-4) x 10(5) M-1.s-1. Substituted isocoumarins are potent inhibitors of proteinase 3 and the best inhibitors are 7-amino-4-chloro-3-(2-bromoethoxy)isocoumarin and 3,4-dichloroisocoumarin (DCI) with kobs/[I] values of 4700 and 2600 M-1.s-1, respectively. Substituted isocoumarins, peptide phosphonates and chloromethyl ketones inhibited proteinase 3 less potently than human neutrophil elastase (HNE) by 1-2 orders of magnitude.     

Vampire bat salivary plasminogen activator exhibits a strict and fastidious requirement for polymeric fibrin as its cofactor, unlike human tissue-type plasminogen activator. A kinetic analysis.

The vampire bat salivary plasminogen activator (BatPA) is virtually inactive toward Glu-plasminogen in the absence of a fibrin-like cofactor, unlike human tissue-type plasminogen activator (tPA) (the kcat/Km values were 4 and 470 M-1 s-1, respectively). In the presence of fibrin II, tPA and BatPA activated Glu-plasminogen with comparable catalytic efficiencies (158,000 and 174,000 M-1 s-1, respectively). BatPA's cofactor requirement was partially satisfied by polymeric fibrin I (54,000 M-1 s-1), but monomeric fibrin I was virtually ineffective (970 M-1 s-1). By comparison, a variety of monomeric and polymeric fibrin-like species markedly enhanced tPA-mediated activation of Glu-plasminogen. Fragment X polymer was 2-fold better but 9-fold worse as cofactor for tPA and BatPA, respectively, relative to fibrin II. Fibrinogen, devoid of plasminogen, was a 10-fold better cofactor for tPA than fibrinogen rigorously depleted of plasminogen, Factor XIII, and fibronectin; the enhanced stimulatory effect of the less-purified fibrinogen was apparently due to the presence of Factor XIII. By contrast, the two fibrinogen preparations were equally poor cofactors of BatPA-mediated activation of Glu-plasminogen. BatPA possessed only 23 and 4% of the catalytic efficiencies of tPA and two-chain tPA, respectively, in hydrolyzing the chromogenic substrate Spectrozyme tPA. However in the presence of fibrin II, BatPA and tPA exhibited similar kcat/Km values for the hydrolysis of Spectrozyme tPA. Our data revealed that BatPA, unlike tPA, displayed a strict and fastidious requirement for polymeric fibrin I or II. Consequently, BatPA may preferentially promote plasmin generation during a narrow temporal window of fibrin formation and dissolution.     

Cadmium(II) complexes of the glycerophosphodiester-degrading enzyme GpdQ and a biomimetic N,O ligand

The glycerophosphodiester-degrading enzyme GpdQ from Enterobacter aerogenes is a promising bioremediator owing to its ability to degrade some organophosphate pesticides and diester products originating from the hydrolysis of nerve agents such as VX. Here, the cadmium derivative of GpdQ was prepared by reconstituting the apoenzyme. Catalytic measurements with (Cd²⁺)₂-GpdQ and the phosphodiester substrate bis(4-nitrophenyl)phosphate yield k _cat = 15 s⁻¹. The pK _a of 9.4, determined from the pH dependence of the catalytic activity, implicates a hydroxide ligand as the catalytic nucleophile. Also prepared was the cadmium-containing biomimetic [Cd₂((HP)₂B)(OAc)₂(OH₂)](PF₆) (where (HP)₂B is [2,6-bis([(2-pyridylmethyl)(2-hydroxyethyl)amino]methyl)-4-methylphenol]), which mimics the asymmetry of the metal ion coordination in the active site of GpdQ. The phosphoesterase-like activity of [Cd₂((HP)₂B)(OAc)₂(OH₂)](PF₆) was studied using the substrate bis(2,4-dinitrophenyl)phosphate, yielding a kinetically relevant pK _a of 8.9, with k _cat = 0.004 s⁻¹. In summary, the model is both an adequate structural and a reasonable functional mimic of GpdQ.     

A survey of the kinetic parameters of class C beta-lactamases. Penicillins.

The interaction between six class C beta-lactamases and various penicillins has been studied. All the enzymes behaved in a very uniform manner. Benzylpenicillin exhibited relatively low kcat. values (14-75 s-1) but low values of Km resulted in high catalytic efficiencies [kcat./Km = 10 X 10(6)-75 X 10(6) M-1.s-1]. The kcat. values for ampicillin were 10-100-fold lower. Carbenicillin, oxacillin cloxacillin and methicillin were very poor substrates, exhibiting kcat. values between 1 x 10(-3) and 0.1 s-1. The Km values were correspondingly small. It could safely be hypothesized that, with all the tested substrates, deacylation was rate-limiting, resulting in acyl-enzyme accumulation.     

Barnase has subsites that give rise to large rate enhancements.

Barnase is found to have a series of subsites for binding its substrates that confers large rate enhancements. Ribonucleotide substrates of the type Zp0Gp1Xp2Y have been synthesized, where p is phosphate, X, Y, and Z are nucleosides, and G is guanosine. G occupies the primary specificity site. The most important subsite is for p2, followed by that for Y. There appears to be no subsite for the Z or p0 positions. Occupation of the subsite for p2 gives rise to a 1000-fold increase in kcat/Km, composed of a 100-fold increase in kcat and a 10-fold decrease in Km. The Y subsite gives rise to further 20-fold increase in kcat/Km. Rates approaching diffusion control for kcat/Km are observed. kcat for the dinucleotide monophosphate GpU = 0.55 s-1, and Km = 240 microM; this compares with 53 s-1 and 20 microM for GpUp, and 3.3 x 10(3) s-1 and 17 microM for GpApA (the best substrate tested). Cleavage occurs at the 3'-phosphate of guanosine in all cases. There are differences in base specificity at the two subsites for X and Y downstream of the scissile bond. The binding energies of different substrates have been analyzed using thermodynamic cycles. These show that the contributions of the X and Y sites are nonadditive.