Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Purification and properties of 4-hydroxycyclohexanecarboxylate dehydrogenase from Corynebacterium cyclohexanicum

4-Hydroxycyclohexanecarboxylate dehydrogenase, which requires NAD as a cofactor, was detected in crude soluble extracts of Corynebacterium cyclohexanicum grown on cyclohexanecarboxylic acid as the sole carbon source. The dehydrogenase was purified from extracts to an electrophoretically homogenous state by ammonium sulfate precipitation and chromatography on DEAE-650s, agarose-NAD and hydroxyapatite. The enzyme consisted of two identical subunits and had a native relative molecular mass of 53,600. There were two residues each of cysteine and tryptophan in the enzyme molecule. Oxo acid rather than hydroxy acid was routinely used as substrate for assay of the enzyme. The enzyme is highly specific for 4-oxocyclohexanecarboxylic acid: the carboxyl group is essential and the position of carbonyl group is important; neither the 2-oxo nor the 3-oxo homologue was used as substrate. A methyl substitution on the ring of 4-oxocyclohexanecarboxylate resulted in an almost complete loss of its activity. The reduction product was identified as trans-4-hydroxycyclohexanecarboxylic acid by gas-liquid chromatography and mass spectrometry. It was used as a substrate for the reverse reaction in the presence of NAD but not its cis-isomer. The enzyme was specific for the B-side (pro-S) hydrogen of NADH in the hydrogen transfer from NADH to 4-oxocyclohexanecarboxylate. The Km values for 4-oxocyclohexanecarboxylate and NADH in the reduction reaction at pH 6.8 were 0.50 mM and 0.28 mM, respectively, whereas those for trans-4-hydroxycyclohexanecarboxylate and NAD in the oxidation reaction at pH 8.8 were 0.51 mM and 0.23 mM, respectively. The equilibrium constant of the reaction was 1.79 x 10(-10) M. The enzyme was strongly inhibited by N-bromosuccinimide.     

Purification and properties of Pichia guilliermondii yeast alkaline nucleotide pyrophosphatase hydrolyzing flavin adenine dinucleotide

Alkaline nucleotide pyrophosphatase was isolated from the Pichia guilliermondii Wickerham ATCC 9058 cell-free extracts. The enzyme was 740-fold purified by saturation of ammonium sulphate, gel-chromatography on Sephadex G-150 and ion-exchange chromatography on DEAE-cellulose. Nucleotide pyrophosphatase is the most active at pH 8.3 and 49 degrees C. The enzyme catalyzes the hydrolysis of FAD, NAD+, NADH, NADPH, GTP. The Km value for FAD is 2.4 x 10(-4) M and for NAD+--5.7 x 10(-6) M. The hydrolysis of FAD was inhibited by NAD+, NADP+, ATP, AMP, GTP, PPi and Pi. The Ki for NAD+, AMP and Na4P2O7 was 1.7 x 10(-4) M, 1.1 x 10(-4) M and 5 x 10(-5) M, respectively. Metal chelating compounds, 8-oxyquinoline, o-phenanthroline and EDTA, inhibited completely the enzyme activity. The EDTA effect was irreversible. The molecular weight of the enzyme determined by gel-filtration on Sephadex G-150 and thin-layer gel-filtration chromatography was 78000 dalton. Protein-bound FAD of glucose oxidase is not hydrolyzed by the alkaline nucleotide pyrophosphatase. The enzyme is stable at 2 degrees C in 0.01 M tris-HCl-buffer (pH 7.5).     

Inhibition of the malic enzyme from Acinetobacter calcoaceticus by NADPH and NADH]

The malic enzyme enriched from Acinetobacter calcoaceticus is inhibited by NADPH and NADH. The inhibition afforded by the reduced coenzymes is not affected by NAD+, AMP and 3'.5'-AMP. Against L-malate, NADPH inhibits the enzyme in a noncompetitive linear fashion (Ki = 1.5 x 10(-4) M), against NADP+, competitively linearly (Ki = 5.0 x 10(-5) M). While NADPH acted as a product inhibitor, NADH seems to be an allosteric effector of the malic enzyme, because with L-malate as the variable substrate in the double reciprocal plot, a nonlinear curve is obtained.     

Evaluation of equilibrium constants for the interaction of lactate dehydrogenase isoenzymes with reduced nicotinamide-adenine dinucleotide by affinity chromatography.

Rabbit muscle lactate dehydrogenase was subjected to frontal affinity chromatography on Sepharose-oxamate in the presence of various concentrations of NADH and sodium phosphate buffer (0.05 M, pH 6.8) containing 0.5 M-NaCl. Quantitative interpretation of the results yields an intrinsic association constant of 9.0 x 10 (4)M-1 for the interaction of enzyme with NADH at 5 degrees C, a value that is confirmed by equilibrium-binding measurements. In a second series of experiments, zonal affinity chromatography of a mouse tissue extract under the same conditions was used to evaluate assoication constants of the order 2 x 10(5)M-1, 3 x 10(5)M-1, 4 x 10(5)M-1, 7 x 10(5)M-1 and 2 x 10(6)M-1 for the interaction of NADH with the M4, M3H, M2H2, MH3 and H4 isoenzymes respectively of lactate dehydrogenase.     

Nicotinamide Adenine Dinucleotide Phosphate-Dependent Formate Dehydrogenase from Clostridium thermoaceticum: Purification and Properties

The nicotinamide adenine dinucleotide phosphate (NADP)-dependent formate dehydrogenase in Clostridium thermoaceticum used, in addition to its natural electron acceptor, methyl and benzyl viologen. The enzyme was purified to a specific activity of 34 (micromoles per minute per milligram of protein) with NADP as electron acceptor. Disc gel electrophoresis of the purified enzyme yielded two major and two minor protein bands, and during centrifugation in sucrose gradients two components of apparent molecular weights of 270,000 and 320,000 were obtained, both having formate dehydrogenase activity. The enzyme preparation catalyzed the reduction of riboflavine 5'-phosphate flavine adenine dinucleotide and methyl viologen by using reduced NADP as a source of electrons. It also had reduced NADP oxidase activity. The enzyme was strongly inhibited by cyanide and ethylenediaminetetraacetic acid. It was also inhibited by hypophosphite, an inhibition that was reversed by formate. Sulfite inhibited the activity with NADP but not with methyl viologen as acceptor. The apparent K(m) at 55 C and pH 7.5 for formate was 2.27 x 10(-4) M with NADP and 0.83 x 10(-4) with methyl viologen as acceptor. The apparent K(m) for NADP was 1.09 x 10(-4) M and for methyl viologen was 2.35 x 10(-3) M. NADP showed substrate inhibition at 5 x 10(-3) M and higher concentrations. With NADP as electron acceptor, the enzyme had a broad pH optimum between 7 and 9.5. The apparent temperature optimum was 85 C. In the absence of substrates, the enzyme was stable at 70 C but was rapidly inactivated at temperatures above 73 C. The enzyme was very sensitive to oxygen but was stabilized by thiol-iron complexes and formate.     

Kinetic studies on microsomal glucose dehydrogenase in rat liver.

Glucose dehydrogenase from rat liver microsomes was found to react not only with glucose as a substrate but also with glucose 6-phosphate, 2-deoxyglucose 6-phosphate and galactose 6-phosphate. The relative maximum activity of this enzyme was 29% for glucose 6-phosphate, 99% for 2-deoxyglucose 6-phosphate, and 25% for galactose 6-phosphate, compared with 100% for glucose with NADP. The enzyme could utilize either NAD or NADP as a coenzyme. Using polyacrylamide gradient gel electrophoresis, we were able to detect several enzymatically active bands by incubation of the gels in a tetrazolium assay mixture. Each band had different Km values for the substrates (3.0 x 10(-5)M glucose 6-phosphate with NADP to 2.4M glucose with NAD) and for coenzymes (1.3 x 10(-6)M NAD with galactose 6-phosphate to 5.9 x 10(-5)M NAD with glucose). Though glucose 6-phosphate and galactose 6-phosphate reacted with glucose dehydrogenase, they inhibited the reaction of this enzyme only when either glucose or 2-deoxyglucose 6-phosphate was used as a substrate. The Ki values for glucose 6-phosphate with glucose as substrate were 4.0 x 10(-6)M with NAD, and 8.4 x 10(-6)M with NADP; for galactose 6-phosphate they were 6.7 x10(-6)M with NAD and 6.0 x 10(-6)M with NADP. The Ki values for glucose 6-phosphate with 2-deoxyglucose 6-phosphate as substrate were 6.3 x 10(-6)M with NAD and 8.9 x 10(-6)M with NADP; and for galactose 6-phosphate, 8.0 x 10(-6)M with NAD and 3.5 x 10(-6)M with NADP. Both NADH and NADPH inhibited glucose dehydrogenase when the corresponding oxidized coenzymes were used (Ki values: 8.0 x 10(-5)M by NADH and 9.1 x 10(-5)M by NADPH), while only NADPH inhibited cytoplasmic glucose 6-phosphate dehydrogenase (Ki: 2.4 x 10(-5)M). The results indicate that glucose dehydrogenase cannot directly oxidize glucose in vivo, but it might play a similar role to glucose 6-phosphate dehydrogenase. The differences in the kinetics of glucose dehydrogenase and glucose 6-phosphate dehydrogenase show that glucose 6-phosphate and galactose 6-phosphate could be metabolized in quite different ways in the microsomes and cytoplasm of rat liver.     

Dimerization of ferulic and caffeic acids by purified peroxidase isolated from Bupleurum salicifolium callus culture

A novel peroxidase that catalyses the transformation of caffeic acid and ferulic acid via oxidative coupling was purified from callus cultures of Bupleurum salicifolium petioles. The enzyme, which was purified over 2,900-fold, is a glycoprotein with a molecular weight of 38,000, determined by SDS/PAGE and gel filtration. The K(m) values obtained were 2.4x10(-4) M for caffeic and 2.6x10(-4) M for ferulic acid, while the K(m) values for H2O2 with caffeic acid was 4x10(-5) M and for H2O2 with ferulic acid was 4.8x10(-4) M. The purified peroxidase exhibits lower activity with typical peroxidase substrates (guaiacol and pyrogallol) than it does with caffeic and ferulic acids, but does not exhibit any activity with other phenylpropanoids tested (cinnamic acid, coumaric acid, and 3,4-dimethoxycinnamic acid).     

The alpha-2 isomer of the sodium pump is inhibited by calcium at physiological levels

The inhibition of the (Na,K)ATPase by calcium was investigated in plasma membrane preparations of rat axolemma, skeletal muscle and kidney outer medulla. Ouabain titration curves demonstrated that physiological calcium (0.08-5 microM) inhibited mainly the high affinity alpha 2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40-50% of the activity with a Ki of 1.9 +/- 0.9 x 10(-7) M. In skeletal muscle high and low ouabain affinity components were present in equal amounts and calcium inhibited only the high affinity component with a Ki of 1.3 +/- 0.3 x 10(-7) M. Kidney enzyme had a low affinity for ouabain and showed very little sensitivity to calcium in the physiological range. It was demonstrated that high calcium levels inhibit the enzyme in a general sense, irrespective of the isomer, with a Ki of 6.5 +/- 6 x 10(-4) M for the kidney and 5.9 +/- 4 x 10(-4) M for the axolemma enzymes. In axolemma, enzyme activity was studied as a function of sodium concentration. Physiological calcium reduced Vmax while not significantly changing K 0.5 for sodium binding.     

D-(+)-lactate dehydrogenase from Lactobacillus murinus

D-(+)-Lactate dehydrogenase from Lactobacillus murinus was purified 670-fold. The Mr was 140,000 as determined by gel filtration. Maximum enzymatic activity was observed at 25 degrees C and pH 6.0 in 200 mM Na2KPO4 buffer. When the temperature was increased from 60 to 65 degrees C, the enzyme was completely inactive in 5 min. The apparent Km for pyruvate and NADH were 4.7 x 10(-4) and 1 x 10(-5) M, respectively. Pyruvate analogs such as oxalate, oxamate, 2-oxobutyrate, and malonate acted as a competitive inhibitors. L-Lactate and L-malate were noncompetitive inhibitors.     

Trypanosoma evansi sialidase: surface localization,properties and hydrolysis of ghost red blood cells and brain cells-implications in trypanosomiasis

A membrane-bound sialidase was isolated from blood stream (BS) Trypanosoma evansi partially purified and characterized. The enzyme is a glycosyl phosphatidyl inositol (GPI) membrane anchored protein. It was solubilized from T. evansi cells recovered from infected camel blood by detergent treatment with Triton CF 54 and partially purified by a series of chromatography steps. The enzyme was optimally active at pH 5.5 and 37 degrees C. It had a KM and Vmax values of 4.8 x 10(-6) M and 3.75 x 10(-6) mol/min x mg protein with Neu5Acalpha2, 3lac as substrate respectively. The KM and Vmax values with fetuin (4-nitrophenyl-oxamic acid) as substrate were 2.9 x 10(-2) M and 4.2 x 10(-3) mol/min x mg protein in the same respect. Kinetic analysis with methly umbelliferyl sialate (MU-Neu5Ac) gave KM and Vmax values of 0.17 mM and 0.84 mmol/min x mg protein respectively. The T. evansi SD could hydrolyse internally linked sialic acid residues of the ganglioside GM2, but was inactive towards colomic acid, and NeuSAc2, 6. lac. When ghost red blood cell (RBC) was used as substrate, it desialylated the RBC in the following order of efficiency; mouse, rat, camel, goat, and dog. Similarly, cerebral cells isolated from BalbC mouse was desialylated by the T. evansi SD. Inhibition studies using 2-deoxy-2, 3 didehydro-N-acetyl neuraminic acid (NeuAc2, 3en) against MU-Neu5Ac revealed a competitive inhibition pattern with Ki of 5.8 microM. The enzyme was also inhibited non-competitively by parahydroxy oxamic acid (pHOA), and competitively by N-ethylmaleimide and N-bromosuccinate with Ki values of 25, 42, and 53 microM, respectively. It was activated by Mg2+ ion and inhibited by Cu2+ and Zn2+.     

Design of potent and specific inhibitors of carboxypeptidases A and B.

The combination in one molecule of functional groups that can interact specifically with different substrate binding areas at the active site of carboxypeptidases A and B has led to the development of potent and specific inhibitors of these enzymes. 2-Benzyl-3-mercaptopropanoic acid (SQ 14,603) has a Ki of 1.1 x 10(-8) M vs. carboxypeptidase A and a Ki of 1.6 x 10(-4) M vs. the B enzyme. 2-Mercaptomethyl-5-guanidinopentanoic acid (SQ 24,798) has a Ki of 4 x 10(-10) M vs. carboxypeptidase B and a Ki of 1.2 x 10(-5) M vs. carboxypeptidase A. It is proposed that the sulfhydryl groups of these inhibitors bind to the catalytically important zinc ions of these enzymes, and that, in conjunction with the benzyl and guanidinopropyl side chains, they are responsible for their specificity.     

Hydroxylating activity of frog epidermis tyrosinase.

Trypsin activated in a similar way both the tyrosine hydroxylase and the dopa-oxidasa activities of frog epidermis tyrosinase. Several electron donors reduced or eliminated the lag period for the hydroxylating enzyme. 4 x 10(-5) M dopa was particularly effective, but without affecting the stationary activity after lag period. Tyrosine hydroxylase had KM = 2.6 X 10(-3) M for tyrosine and 2 x 10(-3) M dopa was a competitive inhibitor with Ki = 5 x 10(-4) M. The enzyme was inactivated during its actuation. Data on thermal denaturation were similar to other obtained from dopa oxidase. Our results tend to confirm our previous hypothesis that the activatory process of the enzyme is accompanied by a spatial unfolding of the enzyme molecule.     

Subcellular distribution and properties of progesterone (delta4-steroid) 5alpha-reductase in rat medial basal hypothalamus.

The subcellular distribution and properties of rat hypothalamic progesterone 5 alpha-reductase, which accelerates the conversion of progesterone to 5 alpha-pregnane-3,20-dione, have been investigated by utilizing 3H-labeled substrate and a reverse isotopic dilution assay system. The enxymic activity was associated primarily with a cell debris-membranes fraction deribed from the 100 x g pellet. This fraction contained mainly membrane-like particulates and was free of nuclei. Little or no activity was associated with the purified nuclei. The hypothalamic 5 alpha-reductase was stimulated by NADPH but not by NADH. The reaction proceeded optimally over a pH range of 6.0 to 7.2 and at a temperaturhe substrate specificity of the enzyme for other delta 4-3-ketosteroids and the ability of these steroids to inhibit the 5 alpha reduction of [1,2-3H]progesterone as well as the effect of 17 beta-estradiol were also studied. 20 alpha-hydroxypregn-4-en-3-one was more reactive that progesterone, while testosterone was the least reactive. The estimated Km for 20 alpha-hydroxypregn-4-en-3-one was 8.6 +/- 1.9 x 10(-7) M, and for testosterone, 1.6 +/- 1.4 x 10(-5) M. The inhibition studies indicate that 20 alpha-hydroxypregn-4-en-3-one and 17 beta-estradiol are competitive and noncompetitive inhibitors, respectively, of the 5 alpha reduction of progesterone with Ki of 6.0 +/- 3.0 x 10(-8) M for 20 alpha-hydroxypregn-4-en-3-one and Kii (intercept inhibition constant) of 2.6 +/- 0.7 x 10(-5) M and Kis (slope inhibition constant) of 3.6 +/- 0.6 x 10(-5) M for 17 beta-estradiol. Testosterone is a poor competitive inhibitor of the reaction.     

Purification and characterization of thermostable beta-N-acetylhexosaminidase of Bacillus stearothermophilus CH-4 isolated from chitin-containing compost.

Thermostable exochitinase was purified to homogeneity from the culture fluid of Bacillus stearothermophilus CH-4, which was isolated from agricultural compost containing shrimp and crabs. The enzyme was a single polypeptide with a molecular mass of 74 kDa, and the N-terminal amino acid sequence was WDKVGVTDLI ISLNIPEADAVVVGMTLQLQALHLY. The enzyme specifically hydrolyzed C-4 beta-anomeric bonding of N-acetylchitooligosaccharides, as well as their p-nitrophenyl (pNP) derivatives. The enzyme also hydrolyzed pNP-beta-N-acetyl-D-galactosaminide (26% of the activity of pNP-beta-N-acetyl-D-glucosaminide). These results indicated that the enzyme is a beta-N-acetylhexosaminidase (EC 3.2.1.52). Kms for acetylchitooligosaccharides were 1 x 10(-4) to 6 x 10(-4) M, while those for the pNP derivatives were 4 x 10(-3) to 8 x 10(-3) M. The optimum temperature of the enzyme was 75 degrees C, and it retained 100 and 28% reactivity after heating at 60 and 80 degrees C, respectively. The enzyme exhibited 15 to 20% activity in a reaction mixture containing 80% organic solvents and maintained 91% of its original activity after exposure to 8 M urea. The optimum and stable pH was around 6.5. Fe2+, Zn2+, and Ca2+ activated the enzyme, but Hg2+ was inhibitory. N-Acetyl-D-glucosamine inhibited the enzyme competitively (Ki = 4.3 x 10(-4) M), whereas N-acetyl-D-galactosamine did not; in contrast, D-glucosamine and D-galactosamine activated it.     

Chloroplast leucyl-tRNA synthetase from Euglena gracilis. Purification, kinetic analysis, and structural characterization

Euglena gracilis chloroplast leucyl-tRNA synthetase was purified to homogeneity by a series of steps including ammonium sulfate precipitation and chromatography on hydroxylapatite, DEAE-cellulose, Sepharose 6B, phosphocellulose, and Blue Dextran-Sepharose. The purified enzyme exhibits a specific activity of 1233 units/mg of protein, which is one of the highest specific activities obtained for an aminoacyl-tRNA synthetase prepared from plant cells. The enzyme has an apparent Km value of 8 x 10(-6) M for L-leucine, 1.3 x 10(-4) M for ATP, and 1.3 x 10(-6) M for tRNALeu. Chloroplast leucyl-tRNA synthetase appears to be a monomeric enzyme with a molecular weight of 100 000. The amino acid composition of chloroplast leucyl-tRNA synthetase has been determined. It is the first reported for a chloroplast aminoacyl-tRNA synthetase, and it reveals a relatively large proportion of apolar residues, as in the case of prokaryotic aminoacyl-tRNA synthetases.     

Purification and properties of inosine monophosphate oxidoreductase from nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp)

Using ammonium sulfate precipitation, gel filtration, and affinity chromatography, inosine monophosphate (IMP) oxidoreductase (EC 1.2.1.14) was isolated from the soluble proteins of the plant cell fraction of nitrogen-fixing nodules of cowpea (Vigna unguiculata L. Walp). The enzyme, purified more than 140-fold with a yield of 11%, was stabilized with glycerol and required a sulfydryl-reducing agent for maximum activity. Gel filtration indicated a molecular weight of 200,000, and sodium dodecyl sulfate-gel electrophoresis a single subunit of 50,000 Da. The final specific activity ranged from 1.1 to 1.5 mumol min-1 mg protein-1. The enzyme had an alkaline pH optimum and showed a high affinity for IMP (Km = 9.1 X 10(-6) M at pH 8.8 and NAD levels above 0.25 mM) and NAD (Km = 18-35 X 10(-6) M at pH 8.8). NAD was the preferred coenzyme, with NADP reduction less than 10% of that with NAD, while molecular oxygen did not serve as an electron acceptor. Intermediates of ureide metabolism (allantoin, allantoic acid, uric acid, inosine, xanthosine, and XMP) did not affect the enzyme, while AMP, GMP, and NADH were inhibitors. GMP inhibition was competitive with a Ki = 60 X 10(-6) M. The purified enzyme was activated by K+ (Km = 1.6 X 10(-3) M) but not by NH+4. The K+ activation was competitively inhibited by Mg2+. The significance of the properties of IMP oxidoreductase for regulation of ureide biosynthesis in legume root nodules is discussed.     

Characterization of yeast homoserine dehydrogenase, an antifungal target: the invariant histidine 309 is important for enzyme integrity

Fungal homoserine dehydrogenase (HSD) is required for the biosynthesis of threonine, isoleucine and methionine from aspartic acid, and is a target for antifungal agents. HSD from the yeast Saccharomyces cerevisiae was overproduced in Escherichia coli and 25 mg of soluble dimeric enzyme was purified per liter of cell culture in two steps. HSD efficiently reduces aspartate semialdehyde to homoserine (Hse) using either NADH or NADPH with kcat/Km in the order of 10(6-7) M(-1) x s(-1) at pH 7.5. The rate constant of the reverse direction (Hse oxidation) was also significant at pH 9.0 (kcat/Km approximately 10(4-5) M(-1) x s(-1)) but was minimal at pH 7.5. Chemical modification of HSD with diethyl pyrocarbonate (DEPC) resulted in a loss of activity that could be obviated by the presence of substrates. UV difference spectra revealed an increase in absorbance at 240 nm for DEPC-modified HSD consistent with the modification of two histidines (His) per subunit. Amino acid sequence alignment of HSD illustrated the conservation of two His residues among HSDs. These residues, His79 and His309, were substituted to alanine (Ala) using site directed mutagenesis. HSD H79A had similar steady state kinetics to wild type, while kcat/Km for HSD H309A decreased by almost two orders of magnitude. The recent determination of the X-ray structure of HSD revealed that His309 is located at the dimer interface [B. DeLaBarre, P.R. Thompson, G.D. Wright, A.M. Berghuis, Nat. Struct. Biol. 7 (2000) 238-244]. The His309Ala mutant enzyme was found in very high molecular weight complexes rather than the expected dimer by analytical gel filtration chromatography analysis. Thus the invariant His309 plays a structural rather than catalytic role in these enzymes.     

Purification and some properties of L-glutamate decarboxylase from human brain

Glutamate decarboxylase (EC 4.1.1.15) from human brain has been purified 8000-fold with respect to the initial homogenate. The molecular weight of the native enzyme was found to be 140000 by electrophoresis on a polyacrylamide gradient gel slab. The presence of a single protein band (Mr 67000) on sodium dodecylsulphate/polyacrylamide gel and the existence of only one N-terminal amino acid suggest that the enzyme consists of two similar if not identical polypeptide chains. The Km of the enzyme at the optimum pH of 6.8 is about 1.3 x 10(-3) M for glutamate and 0.13 x 10(-6) M for pyridoxal phosphate. The analysis of the effects of various inhibitors of mouse brain glutamate decarboxylase on the human enzyme confirms the strong competitive inhibition caused by 3-mercaptopropionic acid (Ki = 2.7 x 10(-6) M) while the Ki values for allylglycine and chloride ion are 1.8 x 10(-2) M and 2.2 x 10(-2) M, respectively.     

Chemical synthesis and kinetic study of the smallest naturally occurring trypsin inhibitor SFTI-1 isolated from sunflower seeds and its analogues

The smallest known naturally occurring trypsin inhibitor SFTI-1 (14 amino acid residues head-to-tail cyclic peptide containing one disulfide bridge) and its two analogues with one cycle each were synthesized by the solid phase method. Their trypsin inhibitory activity was determined as association equilibrium constants (K(a)). Additionally, hydrolysis rates with bovine beta-trypsin were measured. Among all three peptides, the wild SFTI-1 and the analogue with the disulfide bridge only had, within the experimental error, the same activity (the K(a) values 1.1 x 10(10) and 9.9 x 10(9) M(-1), respectively). Both peptides displayed unchanged inhibitory activity up to 6 h. The trypsin inhibitory activity of the analogue with the head-to-tail cycle only was 2.4-fold lower. It was also remarkably faster hydrolyzed (k = 1.1 x 10(-4) mol(peptide) x mol(enzyme)(-1) x s(-1)) upon the incubation with the enzyme than the other two peptides. This indicates that the head-to-tail cyclization is significantly less important than the disulfide bridge for maintaining trypsin inhibitory activity.