Peroxidase catalysed aerobic degradation of 5,6,7,8-tetrahydrobiopterin at physiological pH

The aerobic degradation of 5,6,7,8-tetrahydrobiopterin at neutral pH is catalysed by peroxidase (EC 1.11.1.7) and provides quinonoid 7,8-dihydro(6H)biopterin which readily loses the side chain to yield 7,8-dihydro(3H)pterin. The latter is in equilibrium with trace amounts of 6-hydroxy-5,6,7,8-tetrahydropterin (covalent hydrate) which is irreversibly oxidised to quinonoid 6-hydroxy-7,8-dihydro(6H)pterin, and this finally rearranges to 7,8-dihydroxanthopterin. Spectroscopic evidence (ultraviolet, 1H NMR and 13C NMR) is presented for the reversible addition of water across the 5,6-double bond of 7,8-dihydro(3H)pterin. The intermediate quinonoid 6-hydroxy-7,8-dihydro(6H)pterin is a good substrate for dihydropteridine reductase (EC 1.6.99.7) with a Km of 16.3 microM and kcat of 22.5 s-1. The rate of aerobic degradation (oxidation and loss of the side chain) of natural (6R)-5,6,7,8-tetrahydrobiopterin is several times slower than the rate for the unnatural (6S) isomer. By using a modified assay procedure the kinetic parameters for dihydropteridine reductase are as follows: with (6R)-7,8-dihydro(6H)biopterin Km = 1.3 microM and kcat = 22.8 s-1; with (6S)-7,8-dihydro(6H)biopterin Km = 13.5 microM and kcat = 51.6 s-1; and with (6RS)-7,8-dihydro(6H)neopterin Km = 19.2 microM and kcat = 116 s-1.     

New pteridine substrates for dihydropteridine reductase and horseradish peroxidase.

The oxidation of 4,5-diaminopyrimidin-6(1H)-one, 5,6,7,8-tetrahydropteridin-4(3H)-one, its 6-methyl and cis-6,7-dimethyl derivatives, and 6-methyl- and cis-6-7-dimethyl-5,6,7,8-tetrahydropterins, by horseradish peroxidase/H2O2 is enzymic and follows Michaelis-Menten kinetics, and its Km and kcat. values were determined. This oxidation of 5,6,7,8-tetrahydropterins produces quinonoid dihydropterins of established structure, and they are known to be specific substrates for dihydropteridine reductase. By analogy the peroxidase/H2O2 oxidation of the 5,6,7,8-tetrahydropteridin-4(3H)-ones should produce similar quinonoid dihydro species. The quinonoid species derived from 5,6,7,8-tetrahydropteridin-4(3H)-one and its 6-methyl and cis-6,7-dimethyl derivatives are shown to be viable substrates for human brain dihydropteridine reductase, and apparent Km and Vmax. values are reported.     

Reduced 6,6,8-trimethylpterins. Preparation, properties and enzymic reactivities with dihydropteridine reductase, phenylalanine hydroxylase and tyrosine hydroxylase

The substrates of dihydropteridine reductase (EC 1.6.99.7), quinonoid 7,8-dihydro(6 H)pterins, are unstable and decompose in various ways. In attempting to prepare a more stable substrate, 6,6,8-trimethyl-5,6,7,8-tetrahydro(3 H)pterin was synthesised and the quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin derived from it is extremely stable with a half-life in 0.1 M Tris/HCl (pH 7.6, 25 degrees C) of 33 h. Quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin is not a substrate for dihydropteridine reductase but it is reduced non-enzymically by NADH at a significant rate and it is a weak inhibitor of the enzyme: I50 200 microM, pH 7.6, 25 degrees C when using quinonoid 6-methyl-7,8-dihydro(6 H)pterin as substrate. 6,6,8-Trimethyl-5,6,7,8-tetrahydropterin is a cofactor for phenylalanine hydroxylase (EC 1.14.16.1) with an apparent Km of 0.33 mM, but no cofactor activity could be detected with tyrosine hydroxylase (EC 1.14.16.2). Its phenylalanine hydroxylase activity, together with the enhanced stability of quinonoid 6,6,8-trimethyl-7,8-dihydro(6 H)pterin, suggest that it may have potential for the treatment of variant forms of phenylketonuria.     

The mechanism of the quinone reductase reaction of pig heart lipoamide dehydrogenase.

The relationship between the NADH:lipoamide reductase and NADH:quinone reductase reactions of pig heart lipoamide dehydrogenase (EC 1.6.4.3) was investigated. At pH 7.0 the catalytic constant of the quinone reductase reaction (kcat.) is 70 s-1 and the rate constant of the active-centre reduction by NADH (kcat./Km) is 9.2 x 10(5) M-1.s-1. These constants are almost an order lower than those for the lipoamide reductase reaction. The maximal quinone reductase activity is observed at pH 6.0-5.5. The use of [4(S)-2H]NADH as substrate decreases kcat./Km for the lipoamide reductase reaction and both kcat. and kcat./Km for the quinone reductase reaction. The kcat./Km values for quinones in this case are decreased 1.85-3.0-fold. NAD+ is a more effective inhibitor in the quinone reductase reaction than in the lipoamide reductase reaction. The pattern of inhibition reflects the shift of the reaction equilibrium. Various forms of the four-electron-reduced enzyme are believed to reduce quinones. Simple and 'hybrid ping-pong' mechanisms of this reaction are discussed. The logarithms of kcat./Km for quinones are hyperbolically dependent on their single-electron reduction potentials (E1(7]. A three-step mechanism for a mixed one-electron and two-electron reduction of quinones by lipoamide dehydrogenase is proposed.     

Characterization and nucleotide binding properties of a mutant dihydropteridine reductase containing an aspartate 37-isoleucine replacement.

Kinetic constants for the interaction of NADH and NADPH with native rat dihydropteridine reductase (DHPR) and an Escherichia coli expressed mutant (D-37-I) have been determined. Comparison of kcat and Km values measured employing quinonoid 6,7-dimethyldihydropteridine (q-PtH2) as substrate indicate that the native enzyme has a considerable preference for NADH with an optimum kcat/Km of 12 microM-1 s-1 compared with a figure of 0.25 microM-1 s-1 for NADPH. Although the mutant enzyme still displays an apparent preference for NADH (kcat/Km = 1.2 microM-1 s-1) compared with NADPH (kcat/Km = 0.6 microM-1 s-1), kinetic analysis indicates that NADH and NADPH have comparable stickiness in the D-37-I mutant. The dihydropteridine site is less affected, since the Km for q-PtH2 and K(is) for aminopterin are unchanged and the 14-26-fold synergy seen for aminopterin binding to E.NAD(P)H versus free E is decreased by less than 2-fold in the D-37-I mutant. No significant changes in log kcat and log kcat/Km versus pH profiles for NADH and NADPH were seen for the D-37-I mutant enzyme. However, the mutant enzyme is less stable to proteolytic degradation, to elevated temperature, and to increasing concentrations of urea and salt than the wild type. NADPH provides maximal protection against inactivation in all cases for both the native and D-37-I mutant enzymes. Examination of the rat DHPR sequence shows a typical dinucleotide binding fold with Asp-37 located precisely in the position predicted for the acidic residue that participates in hydrogen bond formation with the 2'-hydroxyl moiety of all known NAD-dependent dehydrogenases. This assignment is consistent with x-ray crystallographic results that localize the aspartate 37 carboxyl within ideal hydrogen bonding distance of the 2'- and 3'-hydroxyl moieties of adenosine ribose in the binary E.NADH complex.     

Physarum polycephalum expresses a dihydropteridine reductase with selectivity for pterin substrates with a 6-(1', 2'-dihydroxypropyl) substitution

Physarum polycephalum is one of few non-animal organisms capable of synthesizing tetrahydrobiopterin from GTP. Here we demonstrate developmentally regulated expression of quinoid dihydropteridine reductase (EC 1.6.99.7), an enzyme required for recycling 6,7-[8H]-dihydrobiopterin. Physarum also expresses phenylalanine-4-hydroxylase activity, an enzyme that depends on dihydropteridine reductase. The 24.4 kDa Physarum dihydropteridine reductase shares 43% amino acid identity with the human protein. A number of residues important for function of the mammalian enzyme are also conserved in the Physarum sequence. In comparison to sheep liver dihydropteridine reductase, purified recombinant Physarum dihydropteridine reductase prefers pterin substrates with a 6-(1', 2'-dihydroxypropyl) group. Our results demonstrate that Physarum synthesizes, utilizes and metabolizes tetrahydrobiopterin in a way hitherto thought to be restricted to the animal kingdom.     

Functionally important residues tyrosine-171 and serine-158 in sepiapterin reductase.

The active site of sepiapterin reductase (SPR), which is a member of the NADP(H)-preferring short-chain dehydrogenase/reductase (SDR) family and acts as the terminal enzyme in the biosynthetic pathway of tetrahydrobiopterin cofactor (BH4), was investigated by truncation and site-directed mutagenesis. The truncation mutants showed that N-terminal and C-terminal residues contribute to bind coenzyme and substrate, respectively. The mutant rSPRA29V showed decreased activity; however, the A-X-L-L-S sequence, which has been reported as a putative pterin binding site, was estimated to preferably work as a component in the region for binding coenzyme rather than substrate. Site-directed mutants of rSPRS158D, rSPRY171V, and rSPRK175I showed low, but significant, activity having similar Km values and kcat/Km values less than 25%, for both sepiapterin and NADPH. Both amino acids Tyr-171 and Ser-158 are located within a similar distance to the carbonyl group of the substrate in the crystal structure of mouse SPR, and the double point mutant rSPRY171V+S158D was indicated to be inactive. These results showed that Ser-158, Tyr-171, and Lys-175 contributed to the catalytic activity of SPR, and both Tyr-171 and Ser-158 are simultaneously necessary on proton transfer to the carbonyl functional groups of substrate.     

Catalytic properties of NADPH-specific dihydropteridine reductase from bovine liver

The catalytic properties of a new type of dihydropteridine reductase, NADPH-specific dihydropteridine reductase [EC 1.6.99.10], from bovine liver, were studied and compared with those of the previously characterized enzyme, NADH-specific dihydropteridine reductase [EC 1.6.99.7]. With quinonoid-dihydro-6-methylpterin, approximate Km values of NADPH-specific dihydropteridine reductase for NADPH and NADH were estimated to be 1.4 micron and 2,900 microns, respectively. The Vmax values were 1.34 mumol/min/mg with NADPH and 1.02 mumol/min/mg with NADPH. With NADPH, the Km values of the enzyme for the quinonoid-dihydro forms of 6-methylpterin and biopterin were 1.4 micron and 6.8 microns, respectively. The enzyme was inhibited by its reaction product, NADP+, in a competitive manner, and the inhibition constant was determined to be 3.2 microns. The enzyme was severely inhibited by L-thyroxine and by 2,6-dichlorophenolindophenol.     

Inactivation of dihydropteridine reductase (human brain) by platinum(II) complexes

Potassium tetrachloroplatinate (K2PtCl4) inactivates dihydropteridine reductase from human brain in a time-dependent and irreversible manner. The inactivation has been followed by measuring enzyme activity and fluorescence changes. The enzyme is completely protected from inactivation by NADH, the pterin cofactor [quinonoid 6-methyl-7,8-dihydro(6H)pterin] and dithiothreitol. Evidence is presented that K2PtCl4 reacts at the active site and that (a) thiol group(s) is involved in, or is masked by, this reaction. K2PtCl4 is a stronger inhibitor of human brain dihydropteridine reductase that cis- and trans-diaminodichloroplatinum, cis-dichloro[ethylenediamine]platinum and K4Fe(CN)6, whereas H2PtCl6 is considerably weaker and (Ph3P)3RhCl is inactive.     

Prostaglandin H synthase kinetics. The effect of substituted phenols on cyclooxygenase activity and the substituent effect on phenolic peroxidatic activity.

A series of p- and m-substituted phenols were examined for their effect on the cyclooxygenase activity of prostaglandin H synthase in 0.1 M phosphate buffer at pH 8.0 and 25.0 +/- 0.1 degrees C. A biphasic response was observed. At low concentrations phenols stimulate, but at higher concentrations inhibit, cyclooxygenase activity. Both enhancement and inhibition are increased by phenolic substituents which are electron-donating, quantified by Hammett sigma constants, and hydrophobic, quantified by Hantsch tau constants. The same series of substituted phenols was also reacted with compound II of prostaglandin H synthase at 4.0 +/- 0.5 degrees C. The compound II data fit the Hammett rho sigma equation; no hydrophobicity factors are required. Phenols inhibit cyclooxygenase activity by interfering with the binding of arachidonic acid to compound I and by competing directly with arachidonic acid as reducing substrates for compound I. Phenols stimulate cyclooxygenase activity by acting as reducing substrates for compound II, thereby accelerating the peroxidatic cycle. Phenols also protect the enzyme from self-catalyzed inactivation, most likely by removing the free radical of prostaglandin G2 by reducing it to prostaglandin G2. Kinetic parameters Km and kcat for cyclooxygenase activity were determined in the presence of phenols. Identical values of Km (15.3 +/- 0.5 mM) and kcat (89 +/- 2 s-1) were obtained regardless of which phenol was employed. Therefore these represent the true Km and kcat values for cyclooxygenase activity.     

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.     

Chemical synthesis and papain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide.

The chemical synthesis of N-alpha-benzyloxycarbonyl-L-lysine p-nitroanilide (Z-Lys-pNA) is described in detail. The pH-dependence of the catalytic parameters kcat,' Km and kcat./Km for the papain-catalysed hydrolysis of Z-Lys-pNA are determined. kcat. and Km are pH-independent between pH 5 and pH 7.42, but the pH-dependence of kcat./Km is bell-shaped, decreasing at high and low pH values with pKa values of 7.97 and 4.40 respectively. The catalytic parameters and their pH-dependence are shown to be similar to those reported for other anilide substrates and it is concluded that the Km value of 0.01 mM previously reported [Angelides & Fink (1979) Biochemistry 18, 2355-2369] is incorrect. The possibility of accumulating a tetrahedral intermediate during the papain-catalysed hydrolysis of Z-Lys-pNA is discussed.     

Acryloyl-CoA reductase from Clostridium propionicum. An enzyme complex of propionyl-CoA dehydrogenase and electron-transferring flavoprotein.

Acryloyl-CoA reductase from Clostridium propionicum catalyses the irreversible NADH-dependent formation of propionyl-CoA from acryloyl-CoA. Purification yielded a heterohexadecameric yellow-greenish enzyme complex [(alpha2betagamma)4; molecular mass 600 +/- 50 kDa] composed of a propionyl-CoA dehydrogenase (alpha2, 2 x 40 kDa) and an electron-transferring flavoprotein (ETF; beta, 38 kDa; gamma, 29 kDa). A flavin content (90% FAD and 10% FMN) of 2.4 mol per alpha2betagamma subcomplex (149 kDa) was determined. A substrate alternative to acryloyl-CoA (Km = 2 +/- 1 microm; kcat = 4.5 s-1 at 100 microm NADH) is 3-buten-2-one (methyl vinyl ketone; Km = 1800 microm; kcat = 29 s-1 at 300 microm NADH). The enzyme complex exhibits acyl-CoA dehydrogenase activity with propionyl-CoA (Km = 50 microm; kcat = 2.0 s-1) or butyryl-CoA (Km = 100 microm; kcat = 3.5 s-1) as electron donor and 200 microm ferricenium hexafluorophosphate as acceptor. The enzyme also catalysed the oxidation of NADH by iodonitrosotetrazolium chloride (diaphorase activity) or by air, which led to the formation of H2O2 (NADH oxidase activity). The N-terminus of the dimeric propionyl-CoA dehydrogenase subunit is similar to those of butyryl-CoA dehydrogenases from several clostridia and related anaerobes (up to 55% sequence identity). The N-termini of the beta and gamma subunits share 40% and 35% sequence identities with those of the A and B subunits of the ETF from Megasphaera elsdenii, respectively, and up to 60% with those of putative ETFs from other anaerobes. Acryloyl-CoA reductase from C. propionicum has been characterized as a soluble enzyme, with kinetic properties perfectly adapted to the requirements of the organism. The enzyme appears not to be involved in anaerobic respiration with NADH or reduced ferredoxin as electron donors. There is no relationship to the trans-2-enoyl-CoA reductases from various organisms or the recently described acryloyl-CoA reductase activity of propionyl-CoA synthase from Chloroflexus aurantiacus.     

Kinetics of hydrolysis of amide and anilide substrates of p-guanidino-L-phenylalanine by bovine and porcine trypsins

The rates of hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalaninamide (Bz-GPA-NH2) and N alpha-substituted p-nitroanilides (pNA) of GPA (benzyloxycarbonyl(Z)-GPA-pNA, benzoyl(Bz)-GPA-pNA and acetyl(Ac)-GPA-pNA) by bovine and porcine trypsins were compared with those of arginine (Arg) substrates. The amide type substrates of GPA were hydrolyzed as fast as those of Arg by the two enzymes with much the same kcat/Km values, though significant differences were found between the kcat and Km values of GPA derivatives and those of Arg derivatives. The kinetic behavior of porcine trypsin toward GPA substrates was almost the same as that of the bovine enzyme. The ratio of the kcat value for Bz-GPA-OEt to that for Bz-GPA-NH2 was much larger than that for the ester to amide substrates of arginine, suggesting that the conformational change of the active site of trypsin induced by a benzene ring in the side chain of Bz-GPA-OEt specifically increases the velocity of the deacylation process of the ester substrate. Remarkably low values of both kcat and Km were found for the tryptic hydrolysis of Z-GPA-pNA and Ac-GPA-pNA, as well as on that of Bz-GPA-pNA (Tsunematsu, H., et al. (1983) J. Biochem. 94, 123-128). Z-GPA-pNA is the best substrate for the two trypsins among the three N alpha-substituted anilide substrates of GPA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of GTP cyclohydrolase I and dihydropteridine reductase in rat pheochromocytoma PC 12 cells

The addition of 8-bromo cyclic AMP, forskolin, theophylline, and 3-isobutyl-1-methylxanthine to the medium of PC 12 cells resulted in an increase in GTP cyclohydrolase I activity, but had no effect on dihydropteridine reductase activity, except theophylline which caused a decrease in dihydropteridine reductase activity at 96 h. GTP cyclohydrolase I activity peaked at 24 h and returned to normal 96 h after drug treatment. Cycloheximide decreased GTP cyclohydrolase I activity at 48 and 96 h, but had little effect on dihydropteridine reductase activity. The addition of reserpine selectively increased only GTP cyclohydrolase I activity. The addition of tetrahydrobiopterin and sepiapterin, however, coordinately inhibited both GTP cyclohydrolase I and dihydropteridine reductase activities. It appears that GTP cyclohydrolase I activity in PC 12 cells is regulated by cyclic AMP stimulation and by end-product inhibition, whereas dihydropteridine reductase activity is only subject to pterin inhibition.     

Purification and properties of NADH-specific dihydropteridine reductase from human erythrocytes

NADH-specific dihydropteridine reductase (EC 1.6.99.7) has been purified from human erythrocytes in essentially homogeneous form. The molecular weight of the enzyme was estimated to be 46,000 by Sephadex G-100 gel filtration. The enzyme reacted with antiserum against NADH-specific dihydropteridine reductase from bovine liver and formed a single immunoprecipitin line in the Ouchterlony double-diffusion system. This precipitin line completely fused with that formed between the human liver enzyme and the antiserum. With use of 5,6,7,8-tetrahydro-6-methylpterin, Km values of the erythrocyte enzyme for NADH and NADPH were determined to be 0.94 and 47 mumol/l, respectively. Vmax values were 58.7 mumol/min/mg with NADH and 6.41 mumol/min/mg with NADPH. The average activity of NADH-specific dihydropteridine reductase of 9 human blood samples from healthy males (20-25 years old) was calculated to be approximately 600 mU/g of hemoglobin, 1.8 mU per 20 microliters of blood, or 1.9 mU per 10(8) erythrocytes.     

Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.     

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.     

NADH-specific dihydropteridine reductase in mastocytoma P-815 cells

NADH-specific dihydropteridine reductase [EC 1.6.99.7] was purified from mouse mastocytoma P-815 cells. Km values for NADH and NADPH were determined to be 1.4 microM and 32 microM, respectively, using tetrahydro-6-methylpterin. Molecular weight was 50,000, and subunit molecular weight was 25,000. The enzymes from P-815 and liver of host mouse (DBA/2) showed similar electrophoretic mobility on polyacrylamide gel. The P-815 enzyme reacted with antiserum against bovine liver NADH-specific dihydropteridine reductase, forming a single precipitin line.     

Interactions of derivatives of guanidinophenylalanine and guanidinophenylglycine with Streptomyces griseus trypsin

The rates of hydrolysis of the ester, amide and anilide substrates of p-guanidino-L-phenylalanine (GPA) by Streptomyces griseus trypsin (S. griseus trypsin) were compared with those of arginine (Arg) substrates. The specificity constant (kcat/km) for the hydrolysis of GPA substrates by the enzyme was 2-3-times lower than that for arginine substrates. The kcat and Km values for the hydrolysis of N alpha-benzoyl-p-guanidino-L-phenylalanine ethyl ester (Bz-GPA-OEt) by S. griseus trypsin are in the same order of magnitude as those of N alpha-benzoyl-L-arginine ethyl ester (Bz-Arg-OEt), although both values for the former when hydrolyzed by bovine trypsin are higher by one order of magnitude than those for the latter. The specificity constant for the hydrolysis of Bz-GPA-OEt by S. griseus trypsin is much higher than that for N alpha-benzoyl-p-guanidino-L-phenylglycine ethyl ester (Bz-GPG-OEt). As with the kinetic behavior of bovine trypsin, low values in Km and kcat were observed for the hydrolysis of amide and anilide substrates of GPA by S. griseus trypsin compared with those of arginine substrates. The rates of hydrolysis of GPA and arginine substrates by S. griseus trypsin are about 2- to 62-times higher than those obtained by bovine trypsin. Substrate activation was observed with S. griseus trypsin in the hydrolysis of Bz-GPA-OEt as well as Bz-Arg-OEt, whereas substrate inhibition was observed in three kinds of N alpha-protected anilide substrates of GPA and arginine. In contrast, no activation by the amide substrate of GPA could be detected with this enzyme.