Interactions between native oat phytochrome and tetrapyrroles

The suggestion, that the increase in the far-UV CD signal of the 124 kDa oat phytochrome upon phototransformation of the Pr to Pfr form is possibly due to the chromophore interaction with the N-terminus segment of the phytochrome protein in the Pfr from (Chai, Y.G., Song, P.S., Cordonnier, M.-M. and Pratt, L.H. (1987) Biochemistry 26, 4947-4952), has been investigated by measuring the circular dichroism in the absence of exogenous tetrapyrrolic chromophores (bilirubin, biliverdin, chlorophyllin and hemin). Open tetrapyrrolic chromophores (bilirubin and biliverdin) did not have any significant effect on the phototransformability of the far-UV CD signal of the phytochrome, whereas closed tetrapyrroles (chlorophyllin and hemin) almost completely blocked the increase in the far-UV CD signal upon Pr to Pfr phototransformation. However, closed tetrapyrroles had no effect on the decrease in the CD signal upon Pfr to Pr photoconversion. Secondary structure analysis showed that the alpha-helix content of both Pr and Pfr forms of phytochrome (with 53 and 56% alpha-helical content, respectively) increased to 62% when a 50-fold molar excess of chlorophyllin was added to them separately. Spectral phototransformation of phytochrome was not affected in the presence of tetrapyrroles, except in the case of hemin. A 50-fold molar mass of hemin caused a significant bleaching of the Pfr form of phytochrome but not that of the Pr form. These results suggest that the chromophore-protein interaction is significantly altered during the phototransformation of phytochrome.     

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.     

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.     

The complete amino-acid sequence and the phylogenetic origin of phycocyanin-645 from the cryptophytan alga Chroomonas sp

The first complete amino-acid sequence of the cryptomonad phycobiliprotein phycocyanin-645 from Chroomonas sp. is presented. The alpha 1-subunit contains 70 amino-acid residues and the alpha 2-subunit 80 residues. In each of the alpha-subunits a green, 697-nm absorbing chromophore is covalently bound to Cys18. Both alpha-subunits contain a high number of charged residues. The phycocyanin-645 beta-subunit consists of 177 amino-acid residues. Two phycocyanobilin chromophores are singly bound to Cys beta 82 and Cys beta 158. A purple cryptoviolin-like chromophore is doubly bound to Cys beta 50 and Cys beta 61. Sequence comparisons revealed that the phycocyanin-645 beta-subunit is closely related to red algal phycoerythrin (73% identical amino-acid residues) and not so close to C-phycocyanin (55% identical amino-acid residues). The phycocyanin-645 alpha-subunits represent a special type of phycobiliprotein and a direct relationship to other phycobiliproteins or any light-harvesting polypeptide-pigment complexes could not be derived by sequence comparisons.     

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.     

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.     

Identification of the dehydroascorbic acid reductase and thioltransferase (Glutaredoxin) activities of bovine erythrocyte glutathione peroxidase

Bovine erythrocyte glutathione (GSH) peroxidase (GPX, EC 1.11.1.9) was examined for GSH-dependent dehydroascorbate (DHA) reductase (EC 1.8.5.1) and thioltransferase (EC 1.8.4.1) activities. Using the direct assay method for GSH-dependent DHA reductase activity, GPX had a kcat (app) of 140 +/- 9 min-1 and specificity constants (kcat/Km(app)) of 5.74 +/- 0.78 x 10(2) M-1s-1 for DHA and 1.18 +/- 0.17 x 10(3) M-1s-1 for GSH based on the monomer Mr of 22,612. Using the coupled assay method for thioltransferase activity, GPX had a kcat (app) of 186 +/- 9 min-1 and specificity constants (app) of 1. 49 +/- 0.14 x 10(3) M-1s-1 for S-sulfocysteine and 1.51 +/- 0.18 x 10(3) M-1s-1 for GSH based on the GPX monomer molecular weight. GPX has a higher specificity constant for S-sulfocysteine than DHA, and both assay systems gave nearly identical specificity constants for GSH. The DHA reductase and thioltransferase activities of GPX adds to the repertoire of functions of this enzyme as an important protector against cellular oxidative stress.     

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.     

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.     

Kinetic studies on surface-mediated activation of bovine factor XII and prekallikrein. Effects of kaolin and high-Mr kininogen on the activation reactions

The kaolin-mediated reciprocal activation of bovine factor XII and prekallikrein was divided into the following two reactions: the activation of factor XII by plasma kallikrein (reaction 1) and the activation of prekallikrein by factor XIIa (reaction 2). The effects of high-Mr kininogen and kaolin surface on the kinetics of these activation reactions were studied. High-Mr kininogen markedly enhanced the rate of reactions 1 and 2 in the presence of kaolin, and the enhancements were highly dependent on the concentrations of the protein cofactor and amount of kaolin surface. For the activation of factor XII by plasma kallikrein (reaction 1), high-Mr kininogen was required when a low concentration of factor XII and kaolin was used. The molar ratio of the protein cofactor to factor XII for optimal activation was found to be approximately 1:1. The apparent Km value and the kcat/Km value for plasma kallikrein on factor XII were calculated to be 4 nM and 5.2 X 10(7) s-1 X M-1, respectively. The activation of prekallikrein by factor XIIa, (reaction 2) proceeded even in the absence of high-Mr kininogen and kaolin. The addition of the protein cofactor and surface to the reaction mixture remarkably accelerated the reaction, and the apparent Km value for factor XIIa on prekallikrein was reduced from 1 microM to 40 nM. Moreover, the kcat/Km value was altered from 7.3 X 10(4) to 1.1 X 10(6) s-1 X M-1). These results suggest that high-Mr kininogen accelerates the surface-mediated activation of factor XII and prekallikrein by enhancing the susceptibility of factor XII to plasma kallikrein, on the one hand, and the affinity of factor XIIa for prekallikrein, on the other hand. Kaolin may play an important role in the concentration and organization of these components on the negatively charged surface.     

Nonessentiality of histidine 291 of Rhodospirillum rubrum ribulose-bisphosphate carboxylase/oxygenase as determined by site-directed mutagenesis

Chemical modification of spinach ribulosebisphosphate carboxylase/oxygenase by diethyl pyrocarbonate led to the conclusion that His-298 is an essential active-site residue (Igarashi, Y., McFadden, B. A., and El-Gul, T. (1985) Biochemistry 24, 3957-3962). From the pH dependence of inactivation, the pKa of His-298 was observed to be approximately 6.8, and it was suggested that this histidine might be the essential base that initiates catalysis (Paech, C. (1985) Biochemistry 24, 3194-3199). To explore further the possible function of His-298, we have used site-directed mutagenesis to replace the corresponding residue of the Rhodospirillum rubrum carboxylase (His-291) with alanine. Assays of extracts of Escherichia coli JM107, harboring either the wild-type or mutant gene in an expression vector, revealed that the mutant protein is approximately 40% as active catalytically as the normal carboxylase. After purification to near homogeneity by immunoaffinity chromatography, the mutant protein was partially characterized with respect to subunit structure, kinetic parameters, and interaction with a transition-state analogue. The purified mutant carboxylase had a kcat of 1.5 s-1 and a kcat/Km of 1.7 X 10(4) M-1 s-1 in contrast to values of 3.6 s-1 and 6 X 10(5) M-1 s-1 for the normal enzyme. The high level of enzyme activity exhibited by the Ala-291 mutant excludes His-291 in the R. rubrum carboxylase (and by inference His-298 in the spinach carboxylase) as a catalytically essential residue.     

Reactions of prostaglandin H synthase in the presence of the stabilizing agents diethyldithiocarbamate and glycerol

Both cyclooxygenase and peroxidase reactions of prostaglandin H synthase were studied in the presence and absence of diethyldithiocarbamate and glycerol at 4 degrees C in phosphate buffer (pH 8.0). Diethyldithiocarbamate reacts with the high oxidation state intermediates of prostaglandin H synthase; it protects the enzyme from bleaching and loss of activity by its ability to act as a reducing agent. For the reaction of diethyldithiocarbamate with compound I, the second-order rate constant k2,app, was found to fall within the range of 5.8 x 10(6) +/- 0.4 x 10(6) M-1.s-1 less than k2,app less than 1.8 x 10(7) +/- 0.1 x 10(7) M-1.s-1. The reaction of diethyldithiocarbamate with compound II showed saturation behavior suggesting enzyme-substrate complex formation, with kcat = 22 +/- 3 s-1, Km = 67 +/- 10 microM, and the second-order rate constant k3,app = 2.0 x 10(5) +/- 0.2 x 10(5) M-1.s-1. In the presence of both diethyldithiocarbamate and 30% glycerol, the parameters for compound II are kcat = 8.8 +/- 0.5 s-1, Km = 49 +/- 7 microM, and k3,app = 1.03 x 10(5) +/- 0.07 x 10(5) M-1.s-1. The spontaneous decay rate constants of compounds I and II (in the absence of diethyldithiocarbamate) are 83 +/- 5 and 0.52 +/- 0.05 s-1, respectively, in the absence of glycerol; in the presence of 30% glycerol they are 78 +/- 5 and 0.33 +/- 0.02 s-1, respectively. Neither cyclooxygenase activity nor the rate constant for compound I formation using 5-phenyl-4-pentenyl-1-hydroperoxide is altered by the presence of diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Catalytic competence of human alpha- and gamma-thrombin in the activation of fibrinogen and factor XIII

Steady-state kinetic parameters were compared for the action of alpha- and gamma-thrombin on the physiologically important thrombin substrates fibrinogen and factor XIII at 37 degrees C, pH 7.4, and 0.14 M NaCl. gamma-Thrombin, an alpha-thrombin derivative proteolytically cleaved at R-B73 and K-B154, was observed to catalyze the release of fibrinopeptide A (FPA) from fibrinogen with a specificity constant (kcat/Km) of 5 X 10(3) M-1 s-1. This value was approximately 2400-fold lower than the specificity constant for the corresponding alpha-thrombin-catalyzed reaction. The low specificity constant was attributed to an increase in Km and a decrease in kcat for gamma-thrombin-catalyzed release of FPA from fibrinogen. Conversion of alpha-thrombin to gamma-thrombin also resulted in an approximately 800-fold reduction in the specificity constant for thrombin-catalyzed release of fibrinopeptide B (FPB) from fibrin I, as well as a loss in discriminatory power. Whereas alpha-thrombin preferentially released FPA from intact fibrinogen, gamma-thrombin released FPA and FPB from intact fibrinogen at similar rates. In contrast to the large difference in specificity constants observed for alpha- and gamma-thrombin catalysis with fibrin(ogen) as substrate, the specificity constant (2.6 X 10(4) M-1 s-1) observed for gamma-thrombin-catalyzed release of activation peptide from factor XIII was only 5-fold lower than the corresponding value for the alpha-thrombin-catalyzed reaction. Additionally, the promotion of factor XIII activation by fibrin characteristic of the alpha-thrombin-catalyzed reaction did not occur in the gamma-thrombin-catalyzed reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monofunctional chorismate mutase from Bacillus subtilis: kinetic and 13C NMR studies on the interactions of the enzyme with its ligands

The interaction of the monofunctional chorismate mutase from Bacillus subtilis with chorismate and prephenate has been studied kinetically and by NMR spectroscopy with 13C specifically labeled substrates. Prephenate dominates the population of enzyme-bound species, and the "off" rate constant (approximately 60 s-1) obtained from line-broadening experiments is close to the value of kcat for chorismate (50 s-1) determined kinetically. The calculated "on" rate constant for prephenate (8 x 10(5) M-1 s-1) is similar to the value of kcat/Km for chorismate (5 x 10(5) M-1 s-1). The kinetic parameters of the Bacillus mutase are remarkably insensitive to pH over a wide range and display no solvent isotope effect. These results suggest that the enzyme-catalyzed reaction may be encounter controlled (slowed from the diffusion limit by some feature of the enzyme's active site) and that kcat for chorismate is determined by the product off rate. There is now no evidence to suggest that the skeletal rearrangement on the enzyme surface occurs by a pathway other than a pericyclic process.     

Cryptomonad biliprotein: Phycocyanin-645 from a Chroomonas species

The properties of phycocyanin-645 from the fresh water cryptomonad Chroomonas spec. were investigated after the pigment was isolated and purified by a combination of differential ammonium sulphate fractionation, gel filtration chromatography and ammonium sulphate gradient elution.Phycocyanin-645 is characterized by absorption maxima at 645 nm, 584 nm, 369 nm, 275 nm and shoulders at 340 nm and 620 nm. The CD spectrum has a negative maximum at 645 nm and a positive maximum at 584 nm with a shoulder at 610 nm.The fluorescence emission spectrum is asymmetrical and shows a maximum at 660 nm and a shoulder at approximately 715 nm. The molecular weight of the native phycocyanin-645, estimated by gel filtration, is 45000 for all multiple pigment forms below.Phycocyanin-645 is heterogenous in charge as revealed by isoelectric focusing with pIs at 7.03, 6.17, 5.75, 5.25 and 4.88, respectively, the main bands lying at pI 7.03 and pI 6.17. This was confirmed by polyacrylamide gel electrophoresis; five pigment components differing in mobility were found. We propose the term multiple pigment forms for these five phycocyanin-645 modifications.Calibrated SDS gel electrophoresis shows phycocyanin-645 to consist of three subunits, two light chains (₁, ₂), having molecular weights of 9200 and 10400, respectively, and one heavy chain (), having a molecular weight of 15500. Suggesting a 1:1:2 ratio between the subunits, the quaternary structure of the pigment molecule is _{1 1}-_{2 1}.Abbreviations PC-645 phycocyanin-645 - C-PC C-phycocyanin - SDS sodium dodecyl sulphate - pI isoelectric point - mol. wt. molecular weight     

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.     

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.     

Characterization of the kinetic pathway for liberation of fibrinopeptides during assembly of fibrin

The time dependence of the release of fibrinopeptides from fibrinogen was studied as a function of the concentration of fibrinogen, thrombin, and Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization. The release of fibrinopeptides during fibrin assembly was shown to be a highly ordered process. Rate constants for individual steps in the formation of fibrin were evaluated at pH 7.4, 37 degrees C, gamma/2 = 0.15. The initial event, thrombin-catalyzed proteolysis at Arg-A alpha 16 to release fibrinopeptide A (kcat/Km = 1.09 X 10(7) M-1s-1) was followed by association of the resulting fibrin I monomers. Association of fibrin I was found to be a reversible process with rate constants of 1 X 10(6) M-1s-1 and 0.064 s-1 for association and dissociation, respectively. Assuming random polymerization of fibrin I monomer, the equilibrium constant for fibrin I association (1.56 X 10(7) M-1) indicates that greater than 80% of the fibrin I protofibrils should contain more than 10 monomeric units at 37 degrees C, pH 7.4, when the fibrin I concentration is 1.0 mg/ml. Association of fibrin I monomers was shown to result in a 6.5-fold increase in the susceptibility of Arg-B beta 14 to thrombin-mediated proteolysis. The 6.5-fold increase in the observed specificity constant from 6.5 X 10(5) M-1s-1 to 4.2 X 10(6) M-1s-1 upon association of fibrin I monomers and the rate constant for fibrin association indicates that most of the fibrinopeptide B is released after association of fibrin I monomers. The interaction between a pair of polymerization sites in fibrin I dimer was found to be weaker than the interaction of fibrin I with Gly-Pro-Arg-Pro and weaker than the interaction of fibrin I with fibrinogen.     

Trinitrobenzoylated poly(D-lysine) as a stimulator of interactions between plasminogen, plasmin, and tissue-type plasminogen activator

Trinitrobenzyl alkylation of poly(D-lysine) provides a novel powerful stimulator of tissue-type plasminogen activator. Its stimulatory effect on plasminogen activation is far greater than that of the original poly(D-lysine), and even surpasses that of fibrin. Its effect on plasmin-catalysed modification of both tissue-type plasminogen activator (t-PA) and native (Glu-1-) plasminogen are also investigated. Cleavage of one-chain t-PA to its two-chain form is monitored by measuring the increase in amidolytic activity which accompanies this transformation. Presupposing apparent first-order reaction kinetics, a theory is developed by which the rate constant, kcat/Km = 1.0 X 10(6) M-1 X s-1 of plasmin cleavage of one-chain t-PA can be calculated. Plasmin-catalysed transformation of 125I-labelled Glu-1- to Lys-77-plasminogen is quantified following separation by polyacrylamide gel electrophoresis at pH 3.2. A rate constant, kcat/Km = 4.4 X 10(3) M-1 X s-1 is obtained for the reaction between plasmin and Glu-1-plasminogen in the presence of 1 mM trans-4-(aminomethyl)cyclohexane-1-carboxylic acid. Both of the above plasmin-catalysed reactions are strongly enhanced by trinitrobenzoylated poly(D-lysine). The mechanism of action of this stimulator is elucidated by studying its binding to both activator and plasmin(ogen), and by direct comparison of the results with measurements of plasminogen activation kinetics in the presence of the stimulator. Binding studies are performed exploiting the observation that an insoluble yellow complex is formed between plasminogen and modified poly(D-lysine). Protein-polymer interactions are also studied with solubilised components in an aqueous two-phase partition system containing dextran and poly(ethylene glycol). The rate enhancement of plasminogen activation is found to be closely correlated to the association of plasminogen to the stimulator. It is proposed that the stimulator effects of this simple polymer on the enzymatic activities of both plasminogen activator and plasmin are brought about by association of the proteinase and its substrate to a common matrix. Similarities between the action of the artificial and the natural stimulator (fibrin) are stressed. These properties of trinitrobenzoylated poly(D-lysine) makes it useful as a model for the study of the regulatory mechanism of the fibrinolytic process at the molecular level.     

Energetics of proline racemase: racemization of unlabeled proline in the unsaturated, saturated, and oversaturated regimes

The interconversion of L- and D-proline catalyzed by proline racemase has been studied. The entire time course of the approach to equilibrium has been followed. After a short time the product concentration is significant, and the reaction runs under reversible conditions. As the total substrate concentration is increased, the system moves from the unsaturated regime into the saturated regime. At very high substrate levels under the reversible conditions used, the rate constant for substrate racemization falls, as the system moves into the "oversaturated" regime. Here, the net rate of the enzyme-catalyzed reaction is limited by the rate of return of the free enzyme from the form that liberates product back to the form that binds substrate. The results are analyzed in terms of the simple mechanism (table; see text) and illustrate the additional information that is available from reactions studied under reversible conditions. In the unsaturated region the value of the second-order rate constant kU (equivalent to kcat/Km) is 9 X 10(5) M-1 s-1 in each direction. In the saturated region, kcat = kcat = 2600 s-1 and Km = 2.9 mM. In the oversaturated region, the rate constant kO is 81 M s-1. The substrate concentration at which unsaturated and saturated terms contribute equally is 2.9 mM, and the substrate concentration at which saturated and oversaturated terms contribute equally is 125 mM.