球形红假单胞菌的D-3-羟基丁酸脱氢酶。该酶在溶液中催化化学平衡时放射性同位素再分布的动力学。

D-3-hydroxybutyrate dehydrogenase from Rhodopseudomonas spheroides. Kinetics of radioisotope redistribution at chemical equilibrium catalysed by the enzyme in solutions.

作者信息

Preuveneers M J, Peacock D, Crook E M, Clark J B, Brocklehurst K

出版信息

Biochem J. 1973 May;133(1):159-64. doi: 10.1042/bj1330159.

DOI:10.1042/bj1330159

PMID:4352836

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1177679/

Abstract

The reversible NAD(+)-linked oxidation of d-3-hydroxybutyrate to acetoacetate in 0.1m-sodium pyrophosphate buffer, pH8.5, at 25.0 degrees C, catalysed by d-3-hydroxybutyrate dehydrogenase (d-3-hydroxybutyrate-NAD(+) oxidoreductase, EC 1.1.1.30), was studied kinetically at chemical equilibrium by monitoring radioisotope redistribution with sodium dl-hydroxy[3-(14)C]butyrate and [4-(3)H]NAD(+)(labelled in the nicotinamide ring). 2. When all substrates are maintained at concentrations approaching saturation (approx. 3-50 times the K(m) values) the first-order rate constant for the enzyme-catalysed interconversion of NAD(+) and NADH is much smaller than that for the enzyme-catalysed interconversion of d-3-hydroxybutyrate and acetoacetate. 3. The rate of interconversion of NAD(+) and NADH increases initially with increasing concentrations of d-3-hydroxybutyrate and acetoacetate (ratio of concentrations maintained constant), passes through a maximum and approaches closely to zero at saturating concentrations of the latter substrates. 4. The rates of interconversion of NAD(+) and NADH and of d-3-hydroxybutyrate and acetoacetate increase with increasing concentration of NAD(+) (up to 66 times its K(m) value) and NADH (up to 180 times its K(m) value) (ratio of the concentrations of the nicotinamide nucleotides maintained constant). 5. These findings support the description of this catalysis as an ordered Bi Bi mechanism with no detectable alternative pathway, in which the interconversion of the central ternary complexes is not rate-limiting, and provide no evidence for the formation of dead-end complexes. 6. The solubility of 2,4-dinitrophenylhydrazine in HCl exhibits an acidity optimum, the maximum solubility at 25.0 degrees C (3.8mg/ml, 19mm) occurring at 2.29m-HCl; in solutions of this acidity acetone 2,4-dinitrophenylhydrazone is relatively insoluble (0.098mg/ml, 0.413mm).

摘要

在pH8.5的0.1m焦磷酸钠缓冲液中，于25.0℃下，由d-3-羟基丁酸脱氢酶（d-3-羟基丁酸-NAD⁺氧化还原酶，EC 1.1.1.30）催化的d-3-羟基丁酸可逆地NAD⁺连接氧化为乙酰乙酸的反应，通过用dl-羟基[3-(¹⁴)C]丁酸钠和[4-(³)H]NAD⁺（在烟酰胺环上标记）监测放射性同位素再分布，在化学平衡状态下进行了动力学研究。2. 当所有底物保持在接近饱和的浓度（约为Kₘ值的3 - 50倍）时，酶催化的NAD⁺和NADH相互转化的一级速率常数远小于酶催化的d-3-羟基丁酸和乙酰乙酸相互转化的一级速率常数。3. NAD⁺和NADH的相互转化速率最初随着d-3-羟基丁酸和乙酰乙酸浓度的增加（浓度比保持恒定）而增加，经过一个最大值，在后者底物饱和浓度时接近零。4. NAD⁺和NADH以及d-3-羟基丁酸和乙酰乙酸的相互转化速率随着NAD⁺（高达其Kₘ值的66倍）和NADH（高达其Kₘ值的180倍）浓度的增加而增加（烟酰胺核苷酸浓度比保持恒定）。5. 这些发现支持将这种催化描述为一种有序的双底物双产物机制，没有可检测到的替代途径，其中中心三元复合物的相互转化不是限速步骤，并且没有提供形成死端复合物的证据。6. 2,4-二硝基苯肼在HCl中的溶解度呈现出最佳酸度，在25.0℃时最大溶解度（3.8mg/ml，19mmol/L）出现在2.29m-HCl中；在这种酸度的溶液中，丙酮2,4-二硝基苯腙相对不溶（0.098mg/ml，0.413mmol/L）。