Biochimica et Biophysica Acta (BBA) - Enzymology最新文献

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli is composed of four subunits of seemingly identical molecular weight (Suzuki, S., Yamaguchi, J. and Tokushige, M. (1973) Biochim. Biophys. Acta 321, 369–381). The subunit arrangement of the enzyme was studied by two distinct methods, cross-linking of subunits with a bifunctional reagent, dimethyl suberimidate, and statistical classification of negatively stained electron microscopic images. In the former method, the densitometric patterns of the cross-linked aspartase were analyzed quantitatively after separating each component by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the results were compared with the theoretical distribution. In the latter method, a number of electron microscopic images were classified into several groups according to their characteristic appearance. The results obtained by these two methods are compatible with the possibility that the enzyme has a tetrameric structure consisting of two pairs of dimers, in which the two pairs of rod-shape subunits meet perpendicularly, being typical of D₂ symmetry.

A specific dl-glycerol-1-phosphatase (glycerol-1-phosphate phosphohydrolase, EC 3.1.3.21) has been identified in the halotolerant alga Duniella salina. The enzyme is highly specific for dl-glycerol 1-phosphate, requires magnesium for activity and has a neutral pH optimum. High sensitivity toward sulfhydryl reagents suggests the existence of a sulfhydryl group in close proximity to the active site. Due to instability the enzyme was only partially purified (40-fold). Activity measurements following polyacrylamide electrophoresis showed the enzyme to have a molecular weight around 86 kdaltons. It is suggested that the enzyme plays a major role in the mechanism of osmoregulation in Dunaliella.

The possible noninvolvement of the carbohydrate moiety of human fibrinogen in the clotting mechanism was examined by eliminating the neutral sugar chains from desialylated fibrinogen by almond glycopeptidase digestion. 40% of the total neutral sugars was removed from the desialylated fibrinogen. The neutral sugars from both the β- and γ-polypeptide chains were released equally. The protein moiety of the glycopeptidase-digested fibrinogen was found to be intact. No significant change was observed in the thrombin time (fibrinogen clottability) of the resultant fibrinogen. The results suggest that the carbohydrate moiety of fibrinogen is not involved in the clotting mechanism. Oligosaccharide was detected in the glycopeptidase digest of desialylated fibrinogen by thin-layer chromatography (TLC), and was found to be identical with those released quantitatively from the peptic digests of β- and γ-polypeptide chains. The structure of the sugar chain was identified tentatively as Gal₂-GlcNAc₂-Man₃-GlcNAc₂, by sequential exoglycosidase digestion and quantitative analysis of carbohydrate components.

New analytical methods were devised to estimate the Michaelis constant (K_m), the maximum velocity (V), the concentration of endogenous substrate (x) and the activity of contaminating enzyme (u) in an impure enzyme reaction with single substrate. In the non-radiometric assay, the linear plot was developed on the basis of Eisenthal-Cornish-Bowden plot [1] by transforming the equation for reaction rate (v) consisting of K_m, V,x,u and the concentration of substrate (S′). To confirm the accuracy of the linear plot, the non-linear fitting method was simultaneously devised in terms of a modification of the method of Cleland [2]. In the radiometric assay, the linear and non-linear kinetic analyses were applied to the equation for the radiometric rate ($\text{v}{}^{\mathrm{\ast }}$), expressed by K_m, V, x and $\text{S}{}^{\mathrm{\ast }}$ (concentration of radioactively labelled substrate) as in the non-radiometric assay. In both assays, the values of K_m, V and x (with u value in the non-radiometric assay) were obtainable at x₁ and x₂ with a known ratio of x₂/x₁. The validity of the above methods was proved by the model experiments with purified enzymes; and the radiometric model experiment offered a good example for a new enzymatic assay method of many substrates. These methods were successfully applied to the practical experiments.

The regulatory properties of the lysine-sensitive aspartokinase (ATP : l-aspartate 4-phosphotransferase, EC 2.7.2.4) have been studied under equilibrium conditions by determining the effects of modifiers on the rate of equilibrium isotope exchange between ADP and ATP. The extent of inhibition by lysine, leucine or phenylalanine is almost independent of substrate concentration but is influenced by the substrate/product ratio. Inhibition by a given concentration of inhibitor is increased when the ADP/ATP ratio is increased indicating a regulatory interaction between end products and cellular energy metabolism. Lysine inhibition is cooperative under equilibrium conditions and the parameters of the Hill equation are nearly identical to those obtained in initial velocity studies. A cooperative heterotropic interaction between lysine and leusine is also observed by the ATP-ADP exchange assay just as it is in initial velocity assays. Thus, the regulatory features of aspartokinase that are observed in initial velocity studies are also manifest under equilibrium conditions as revealed by equilibrium isotope exchange rates.

The substrate specificity of proteinase B (EC 3.4.22.9) from Baker's yeast was studied. Experiments with unblocked synthetic peptides indicated that the enzyme has no aminopeptidase activity. The proteinase cleaves trypsin substrates like Bz-Arg-OEt, Bz-Arg-pNA and Bz-Ile-Glu-Gly-Arg-pNA and chymotrypsin substrates like Ac-Tyr-OEt and Bz-Tyr-pNA. The K_m value for Ac-Tyr-OEt is similar to that of chymotrypsin A, but the catalytic activity per mol proteinase B amounts to only $\text{1}\text{20}$ that of chymotrypsin A. K_m and k_cat for Bz-Arg-OEt are $\text{1}\text{50}$ and $\text{1}\text{7}$ as high as the corresponding values determined for trypsin. Proteinase B cleaved the oxidized insulin B chain with an initial rapid cleavage step at Leu(15)-Tyr(16) and Phe(24)-Phe(25). Slower hydrolysis was observed at Gln(4)-His(5), Leu(11)-Val(12) Tyr(16)-Leu(17), Leu(17)-Val(18), Arg(22)-Gly(23) and Phe(25)-Tyr(26). These results suggest that the specificity of proteinase B is comparable to the specificity of porcine chymotrypsin C as well as of trypsin. When the hexapeptide Leu-Trp-Met-Arg-Phe-Ala was used as a substrate for proteinase B, the enzyme preferentially attacked at Arg-Phe and more slowly at Trp-Met.

The molybdoprotein NADH-nitrate reductase (NADH : nitrate oxidoreductase, EC 1.6.6.1) from spinach can be inactivated by acetylene only when the enzyme is in its reduced state. Other gases such as ethylene, carbon monoxide, dinitrogen and others did not alter the enzyme activity. From the two partial activities of nitrate reductase, only the terminal nitrate reductase was impaired by acetylene while the dehydrogenase activity was rather stimulated. Functional dehydrogenase activity was required for inactivation when NADH was the reductant. Dithionite, dithionite + MV or dithionite + FMN were also able to sustain acetylene inactivation, whether or not nitrate reductase was previously depleted of its dehydrogenase activity. However, ascorbate or ascorbate + DCIP did not cooperate with acetylene for inactivating nitrate reductase. Nitrate and the competitive inhibitors with respect to nitrate of nitrate reductase, namely azide, cyanate and carbamyl phosphate, protected nitrate reductase from acetylene inactivation. Cyanide-inactivated nitrate reductase was still sensitive to acetylene, since, once the cyanide-inactivated enzyme was placed under acetylene, no ferricyanide reactivation could be attained. These results suggest that reduced nitrate reductase might bind acetylene at the nitrate active site, where molybdenum is supposed to be implicated, thus impairing the reduction of nitrate.

The binding of dimers of nicotinamide adenine dinucleotide, (NAD)₂, to lactate, malate and alcohol dehydrogenase has been studied by the fluorescence quenching technique. While the alcohol dehydrogenase shows a low binding ability, malate and lactate dehydrogenases have been found to bind (NAD)₂ in a specific way with high affinity. Malate dehydrogenase binds (NAD)₂ more than NADH. All three dehydrogenases are inhibited by (NAD)₂, which behaves as a competitive inhibitor with respect to both NAD⁺ and NADH. These results show that (NAD)₂ is bound to the nucleotide-specific binding site of the dehydrogenases. (NAD)₂ was found to stoichiometrically react with ferricyanide at variance with NADH. The specific interactions with the NAD-dependent dehydrogenases and the ability to enter in monoelectronic redox cycles suggest possible physiological roles for (NAD)₂.

1. Incubation of prolyl 4-hydroxylase (prolyl-glycyl-peptide, 2-oxoglutarate : oxygen oxidoreductase (4-hydroxylating), EC 1.14.11.2) with H₂O₂ leads to a decrease of 50% in the specific activity of enzyme tetramers, followed by dissociation into inactive dimers in which the monomers are covalently cross-linked by S-S bridge formation. 2. Incubation of the enzyme with K₃Fe(CN)₆ leads to a comparable decrease in activity of enzyme tetramers. Addition of urea leads to dissociation into inactive dimers with similarly cross-linked monomers. 3. Removal of the dissociating agent leads to reassociation of cross-linked dimers to tetramers and to about 50% reactivation. The enzyme is further reactivated by preincubation with dithiothreitol. 4. Dissociation of the enzyme with dithiothreitol, urea or LiCl, or at low pH (4.15) produces inactive monomers, which could not be reassociated.

Prenyltransferase (dimethylallydiphosphate : isopentenyldiphosphate dimethylallytransferase, EC 2.5.1.1) has been purified to homogeneity from human liver obtained at autopsy. The enzyme is a dimer with a native molecular weight of 74 000 ± 1 400. The amino acid composition is reported. The enzyme has a broad pH optimum between 7.3 and 8.8 and an absolute requirement for either Mn²⁺ or Mg²⁺ for activity; half-maximal activity was observed at 3.7 μM Mn²⁺ or 89.0 μM Mg²⁺. Michaelis constants for geranyl pyrophosphate and isopentenyl pyrophosphate were 0.44 and 0.94 μM, respectively; the V value for synthesis of farnesyl pyrophosphate from these substrates was 1.1 μmol · min⁻¹ · mg⁻¹. Isopentenyl pyrophosphate inhibited the reaction rates at concentrations above 2 μM when the concentrations of geranyl pyrophosphate were less than 2 μM. The highest concentration of geranyl pyrophosphate tested, 16 μM, showed no inhibition of reaction rates even when the concentration of isopentenyl pyrophosphate was as low as 0.2 μM. Only one form of human liver prenyltransferase could be observed under conditions which resolved the porcine enzyme into two distinct forms; the human enzyme is akin, physico-chemically, to the B-form of the pig liver enzyme. After dialysis against Tris-HCl buffer, pH 7.8, the enzyme became completely dependent upon dithiols or thiols for its activity. Kinetic experiments with a partially activated enzyme sample showed that the activation by the dithiol greatly enhanced the affinity of the enzyme for geranyl pyrophosphate, but not that for isopentenyl pyrophosphate. The human prenyltransferase is inactivated by phenylglyoxal according to pseudo-first-order kinetics, but is protected against the inactivation by 3,3-dimethylallyl and geranyl pyrophosphate. It is also inactivated by high concentrations (>2 mM) of iodoacetic acid, but is protected against the inactivation by dithiothreitol. Antibodies raised to the B-form of the pig liver enzyme cross-reacted with the human prenyltransferase and were 47% as effective in precipitating the human enzyme as the porcine enzyme. In double immunodiffusion experiments the antiserum was monospecific against the B-form of the porcine enzyme; it also gave a single precipitin line with the A-form, but not identical with that given by the B-form. It gave a precipitin line also with the human enzyme, but not identical with that given by either the A- or B-form of the porcine enzyme.