Keitaro Hiromi

Test reactions for a stopped-flow apparatus: Reduction of 2,6-dichlorophenolindophenol and potassium ferricyanide by l-ascorbic acid

Abstract A reaction system suitable for testing the function of a stopped-flow apparatus of high performance was searched for. The reduction of 2,6-dichlorophenol-indophenol by l -ascorbic acid at pH 2.0 was recommended as the most practical test reaction. In due course of the study the reaction mechanism of the reduction was discussed.

Subsite affinities of glucoamylase: Examination of the validity of the subsite theory

Abstract 1. 1. The dependence of Michaelis constant ( K m and the molecular activity ( k 0 ) on the degree of polymerization ( n ) of maltooligosaccharides for the hydrolysis catalyzed by glucoamylase of Rhizopus delemar (α-1,4:1,6-glucan 4:6-glucohydrolase, EC 3.2.1.3) was studied at pH 4.50 and 25 °C, for a range of n from 2 to 15.5. 2. 2. The results were analyzed to evaluate the subsite affinities ( A i ) and the intrinsic rate constant of hydrolysis of substrate linkage in a productive complex ( k int ) according to the theory formerly developed by one of the authors (Hiromi, K. (1970) Biochem. Biophys. Res. Commun. 40, 1–6), which assumed the independency of k int on n and the additivity of A i . 3. 3. The values of K m and k 0 calculated with the above determined values of A i and k int were in excellent agreement with the experimentally obtained ones for n = 2–7 , which confirmed the validity of the theory.

Interpretation of dependency of rate parameters on the degree of polymerization of substrate in enzyme-catalyzed reactions. Evaluation of subsite affinities of exo-enzyme

Abstract The dependency of maximal velocity and Michaelis constant of polymer-degrading enzymes on the degree of polymerization (DP) was interpreted in terms of the subsite affinities by assuming that the intrinsic rate of hydrolysis in the productive complex is independent of DP. The apparent dependency of maximal velocity on DP arises merely from the probability of productive complex formation, which is determined by the arrangement of subsite affinities in the active site of the enzyme. The theory was applied to exo-enzyme, and the method for determining the subsite affinities from the rate parameters was proposed.

Subsite structure and ligand binding mechanism of glucoamylase.

Summary1.The basic concept and outline of the subsite theory were described, which correlates quantitatively the subsite structure (the arrangement of subsite affinities) to the action pattern of amylases in a unified manner.2.The subsite structures of several amylases including glucoamylase were summarized.3.In parallel with the theoretical prediction obtained therefrom, the binding subsites of glucose, gluconolactone and linear substrates to Rhizopus glucoamylase were investigated experimentally, by using steady-state inhibition kinetics, difference absorption spectrophotometry, and fluorometric titration.4.From several lines of evidence, it was concluded that gluconolactone, a transition state analogue, is bound at Subsite I (nonreducing end side) where a tryptophan residue is located.5.The stopped-flow kinetic studies have revealed that all the ligand bindings studied consist of two-step mechanism in which a bimolecular association between the enzyme and a ligand to form a loosely bound complex (EL) followed by the unimolecular isomerization process in which EL converts to the final firmly bound EL* complex. For substrates the EL* may be the productive complex and the fluorescence of the tryptophan located at Subsite 1 is quenched in their isomerization process, most probably a relocation of ligand to occupy this subsite.

In vitro action of human and porcine α-amylases on cyclomalto-oligosaccharides

Abstract The vitro action of human and porcine pancreatic α-amylases on cyclomalto-oligosaccharides (cyclodextrins) was investigated both by a high-performance liquid chromatographic analysis and a quantitative analysis of the reducing power of cyclodextrin hydrolyzates. Cyclomalto-octaose (γ-cyclodextrin) was hydrolyzed to produce mainly maltose, but cyclomalto-hexaose and -heptaose were little affected both by human and porcine α-amylases. Quantitative analysis of reducing power revealed that the ring-opening rate of γ-cyclodextrin catalyzed by human pancreatic α-amylase was 2.8 times slower than that catalyzed by the porcine enzyme. The number of multiple attacks on γ-cyclodextrin and its inhibitor constants for human pancreatic α-amylase and porcine pancreatic α-amylase were almost the same.

Kinetic study of isomerization of ferricytochrome c at alkaline pH

Kinetic studies of the isomerization reaction of horse heart ferricytochrome c between pH 8.5 and pH 12.1 have been carried out by using stopped-flow and rapid scanning stopped-flow techniques. Below pH 10, our results were in good agreement with the scheme proposed earlier (Davis, L. A., Schejter, A. and Hess, G. P. (1974) J. Biol. Chem. 249, 2624–2632). Above pH 10, another faster first-order process was observed, which suggested the existence of a transient species in the isomerization reaction between the species with and without a 695 nm band. The probable scheme of the isomerization reaction is considered to be where H denotes a proton, the colored forms are the species predominant at neutral pH with a 695 nm band and the noncolored forms are the species without a 695 nm band. The transient species has a small 695 nm absorbance which suggests that the sixth ligand is still Met-80, although the protein conformation might be different from that at neutral pH.

A rapid sensitive method for the determination of ascorbic acid in the excess of 2,6-dichlorophenolindophenol using a stopped-flow apparatus

Abstract A rapid and sensitive method termed “time difference analysis” for the determination of reduced ascorbic acid even in the presence of excess triose reductone has been developed by using a stopped-flow apparatus in excess 2,6-dichlorophenolindophenol. The lowest limit of the concentration of ascorbic acid was about 2 × 10−7 m . A single stopped-flow trace can be used for both qualitative and quantitative analysis of ascorbic acid. The contamination of triose reductone, which disturbs the analysis in the ordinary static measurement, can be safely distinguished because of the sluggishness of the reaction.

Kinetics studies on the interactin of rhizopus glucoamylase with maltodextrin and maltotriose, utilizing the absorbance change near 300 nm☆

Abstract MaltodExtrin (high-d.p. malto-oligosaccharides) was found to produce a trough at 303 nm in the difference spectrum of glucoamylase (E.C. 3.2.1.3) from Rhizopus niveus upon binding with the enzyme; this trough disappears upon hydrolysis. The trough, which was ascribed to a change, in the electrostatic environment of a tryptophan residue at the terminal subsite of the enzyme, was found closely related to the formation of the enzyme-substrate complex. The kinetics of binding of maltodextrin and maltotriose to the enzyme were studied at pH 4.5. and 5°, by monitoring the trough by the stopped-flow method. The result was consistent with a two-step mechanism, in which a fast, bimolecular association is followed by a slower, uni-molecular isomerization-process. The latter process involves an environmental change of the tryptophan residue, and is considered to be closely connected to the formation of the productive complex essential for the catalysis.

Analysis of mixtures of α- and β-cyclodextrins using fluorescent dyes

Abstract The fluorescence intensity of 2- p -toluidinylnaphthalene-6-sulphonate (TNS) is markedly increased by the addition of cyclodextrins. Mixtures of α- and β-cyclodextrins can be analysed by utilizing the difference in the sensitivity of fluorescent dyes for the two cyclodextrins. One method utilizes the fluorescence increment of TNS and the fluorescence decrement of o -anisaldehyde produced on binding with cyclodextrins. An alternative method relates the increase in TNS fluorescence to the total amount of cyclodextrins, which is determined from the reducing power after complete hydrolysis by Taka-amylase A (EC 3.2.1.1) and glucoamylase (EC 3.2.1.3).

Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.