Kinetic studies on glucoamylase of rabbit small intestine.

The kinetic properties of a maltase-glucoamylase complex with a neutral pH optimum, purified to homogeneity from the brush borders of the rabbit small intestine, are described. It has a broad range of substrate specificity, hydrolysing di- and poly-saccharides with alpha-1,4 and alpha-1,6 linkages. The Km and Vmax, values of the enzyme for the various substrates were determined. Starch and maltose were its best substrates. The kinetics of hydrolysis of two synthetic linear maltosaccharides, namely maltotriose and maltopentaose, were studied. Mixed-substrate incubation studies revealed the presence of at least two interacting sites on the enzyme, and the data were further analysed by the use of a number of non-substrate inhibitors.     

Mechanism of hydrolyses of phenyl alpha-maltosides catalyzed by taka-amylase A.

Michaelis constants (Kms) and molecular activities (kos) of phenyl, p-nitrophenyl and p-methylphenyl alpha-maltoside for taka-amylase A catalyzed hydrolyses were determined in H2O and in D2O at pH or pD 5.3 and at 25 degrees C. Production of alpha-maltose in the hydrolysis was confirmed by 1H NMR. Neither substituent nor solvent deuterium isotope effects on Kms for phenyl, p-nitrophenyl and p-methylphenyl alpha-maltosides were detected. On the other hand, substituent effects on kos of these compounds were evident, but the isotope effects on kos were not marked, so that protonation of the substrate in the catalytic reaction might not be rate-limiting. The result indicates that nucleophilic attack of a carboxylate anion of the enzyme upon the protonated substrate is the rate-limiting step in the hydrolysis proceeding through the nucleophilic double displacement mechanism, which involves a covalently bonded glycosyl intermediate. The molecular orbitals of phenyl alpha-D-glucosides as model compounds of phenyl alpha-maltosides were calculated by the AM1 method. From the results, it was concluded that the lowering of the lowest unoccupied molecular orbital (LUMO) energy levels and the increase of distribution of LUMO on the anomeric carbon, C-1, of the compounds are caused by protonation at the glycosidic oxygen from the protonated carboxyl group of the enzyme. This causes acceleration of the hydrolysis of a substrate by the enzyme.     

Quantitative determination of anomeric forms of sugar produced by amylases. V. Anomeric forms of maltose produced in the hydrolytic reaction of substituted phenyl alpha-maltosides catalyzed by saccharifying alpha-amylase from B. subtilis.

1. Hydrolyses of phenyl alpha-maltoside and its derivatives with various substituents (p-NO2, p-C1, p-CH3, p-C2H5, and p-C(CH3)3) catalyzed by saccharifying alpha-amylase from B. subtilis3 [EC 3.2.1.1] were studied under conditions such that the products were only maltose and the corresponding phenols (1), in order to determine quantitatively the anomeric form of the sugar produced from each substrate. 2. At the optimum pH of this enzyme (pH-5.4), maltose released from all the substituted substrates studied was entirely in the beta-form. These results are in remarkable contrast to the previous finding that alpha-maltose is exclusively produced from unsubstituted phenyl alpha-maltoside by this enzyme (2). 3. At pH 6.18 and 6.73, maltose produced from unsubstituted phenyl alpha-maltoside (?M) or p-tert-butylphenyl alpha-maltoside (PTB?M) was a mixture of alpha- and beta-anomers, the ratio being dependent on pH as follows: For ?M, the percentage of alpha-anomer was 100% (pH 5.4), 80 (pH 6.18), and 55% (pH 6.73), whereas for PTB?M, the percentage of beta-anomer was 100% (pH 5.4), 75% (pH 6.18), and 60% (pH 6.73).     

Kinetic studies on soluble and insoluble urokinases.

A water-insoluble urokinase (ins-UK) was prepared by covalent coupling to an electrostatically neutral polyacrylamide derivative. The esteratic activity retained by the bound enzyme is about 70 percent of that of the soluble urokinase (UK). Comparative kinetic studies of these two forms of the enzyme were undertaken on lysine esters: N-alpha-acetyl-L-lysine-methyl ester (ALEe) and N-alpha acetylglycyl-L-lysine methyl ester (AGLMe). It was first observed that these substrates both exhibit a marked inhibitory effect toward soluble UK, whereas this phenomenon was less manifest with the insoluble form of the enzyme. Michaelis constants and maximal velocities measured at 33 degrees C, for UK and ins-UK, were identical when ALMe was used, but slightly different with AGLMe. Determination of initial velocities, at a series of pH values shows only minimal differences in the behavior of the soluble enzyme with respect to that of the insoluble form. However, over a range of temperatures, differing Km values for these two enzyme forms were obtained using AGLMe as the substrate. These last results suggest possible interactions between the substrate and the insoluble carrier of the enzyme.     

Kinetic studies on insoluble cellulose-cellulase system.

Enzymatic hydrolysis of insoluble amorphous cellulose by Trichoderma viride cellulase was investigated in a batch reactor at several substrate concentrations and three enzyme levels. The reactions were carried out at 50 degrees C and pH 4.8. Enzyme was rapidly adsorbed onto solids on contact, then gradually returned to the liquid phase as the reaction proceeded. A kinetic model that considered the fast adsorption which was followed by the slow reaction, and subsequent product inhibition was developed to interpret the experimental observations. The resulting equation successfully correlated the data for up to 70% conversion. The methods for determining the kinetic parameters are discussed.     

Kinetic studies on human cysteinyl-tRNA synthetase.

The detailed pH and temperature kinetics of human term placenta cysteinyl-tRNA synthetase (EC 6.1.1.16) were studied. The ATP-PPi exchange reaction catalyzed by the cysteinyl-tRNA synthetase was highly dependent on temperature, pH, and ionic strength. The Arrhenius plot at temperatures between 5 degrees and 40 degrees was linear, giving an activation energy of 19 +/- 2.5 Kcal/mol. The pH dependence of the kinetic parameters Km and Vmax was investigated. Apparent pKa value of 6.4 was observed in the pH-dependence of Vmax/Km plot. The pH versus Vmax plot showed two apparent pKa values of about 5.8 and 7.8. Van't Hoff's enthalpies were used to differentiate the nature of the possible groups responsible for the ionization. These results are valuable for the selection of chemical modifying reagents in characterizing the amino acid residues involved in substrate (nucleotide) binding or catalysis.     

Kinetic studies of drug-dinucleotide complexes.

Three classes of kinetic behavior are observed in the complexes of actinomycin or ethidium with deoxydinucleotides. First, the initial dinucleotide binding to form a 1:1 complex is a rapid bimolecular process, whose rate could be measured for combination of actinomycin with d(pTpG) d(pGpT), d(pGpA), d(pGpG) d(pCpGpG), and d(pCpG) andfor combination of ethidium with d(pGpC). Second, with one exception, all reactions in which a second dinucleotide is added to form a 2:1 dinucleotide-drug complex are limited by a first-order step at high concentration. This class includes the combination of actinomycin with all dinucleotides tested except d(pGpC), and the reaction of ethidium with nucleotides of complementary sequence pyrimidine-purine, such as d(pCpG). The final class is the special case of d(pGpC) interacting to form a 2:1 complex with actinomycin. Third-order kinetics is observed, with no evidence for a first-order, rate-limiting step.     

Steady-state inhibitory kinetic studies on the ligand binding modes of Aspergillus niger glucoamylase.

Inhibitory activities of 1-deoxynojirimycin and gluconolactone on Aspergillus niger glucoamylase were studied in relation to the subsite structure of the enzyme. Although both of these inhibitors are considered to bind at subsite 1 of the enzyme active site, 1-deoxynojirimycin showed competitive type inhibition but gluconolactone was a mixed type (or noncompetitive type) inhibitor for the hydrolysis of p-nitrophenyl alpha-D-glucoside. The former type of inhibition suggested that the main binding mode of the substrate was productive, but the latter, nonproductive. A possible way of explaining these apparent inconsistent results is to assume that the main binding mode of the substrate is productive and gluconolactone forms a nonproductive ternary complex with the enzyme and the substrate.     

Kinetic studies of benzpyrene and hydroxybenzpyrene metabolism.

The kinetics of benzypyrene (BP) metabolism were examined in liver microsomes, and in accordance with the results of Hansen and Fouts [9] exhibited curcilinear Lineweaver-Burk plots. The problem was exacerbated in microsomes of 3-methylcholanthrene (MC-ms) treated rats. The Km for BP, measuring hydroxybenzypyrene (OHBP) appearance was about 0.3 muM in MC-treated adult rats and about 1.0 muM in untreated rats. These values were obtained using a substrate range of 0.2-2.0 muM benzpyrene, 20 mug of microsomal protein/ml and a 3 min assay time. With longer assay times and with higher microsomal protein concentrations curvilinear reciprocal plots were obtained. This was found to be due to a combination of three factors, namely non-specific binding of BP to the microsomes, rapid depletion of substrate, and further metabolism of hydroxy products. At 100 mug microsomal protein/ml about 50% of added BP was non-specifically bound to the microsomes in the range of 0.2-2.0 muM BP. Addition of albumin to the medium (1 mg/ml) greatly enhanced the BP hydroxylase activity but only slightly increased the amount of BP remaining in the medium after sedimentation of the microsomes by centrifugation. 3-OHBP, one of the phenolic products of BP metabolism was found to be metabolized to a non-fluorescent products(s); the Km for this compound was similar to that for BP. Differences were seen in the Vmax rates of BP disappearance and OHBP appearance. Disappearance of BP is several fold faster than OHBP appearance and has a larger Km. The latter may be due to the need to use higher amounts of protein and to allow depletion of enough substrate to make measurements significantly reproducible or the higher Km may reflect a composite value for different routes of BP metabolism.