Michael R. Hollaway

The significant rôle of fructose-1,6-diphosphate in the regulatory kinetics of phosphofructokinase

It is generally accepted that phosphofructokinase (EC 2.7.1.11, here represented as PFK) plays a key role in the regulation of glycolytic flux (for a review see ref. [ 11). Therefore a number of different ligands that influence PFK activity, including the inhibitors ATP and citrate, and the activators AMP, F6P and FDP, represent candidates for the modulation in vivo of the rate of glycolysis (see ref. 121). The present study is concerned mainly with the mechanism for FDP ‘activation’ of PFK. We follow the time-course of the reaction by a method avoiding the use of coupling enzymes and with enzyme concentrations approaching that in vivo. The results indicate a crucial role for FDP in activating PFK under conditions simulating those in vivo, either by increasing the rate of hysteretic transitions, or by inducing a conformational change in a given enzyme molecule to give an increased catalytic activity at the other, non FDPliganded sites.

The kinetics of the reactions of low spin ferric haem undecapeptide with hydrogen peroxide

The kinetics of the reaction of low spin ferric haem undecapeptide (HUPIII) with H2O2 at pH 7.0 and 10.4 at 22 °C have been investigated by stopped flow spectroscopy at 398 nm and analysed by non-linear numerical integration and optimization techniques. At pH 7.0, the only reaction scheme which is found to satisfy the triple requirement of a standard deviation within the standard error of the data, good determination of the omptimized parameters and a random distribution of residuals is a four-step mechanism stated as follows: HUPIIIC + H2O2 → IC → IIC → IIIC, IIIC + H2O2 → IVC at pH 10.4 the only scheme which is found to satisfy the above triple requirement is condensed version of the mechanism at pH 7.0 and can be represented as: HUPIIID + H2O2 → ID → IIID, IIID + H2O2 → IVD The rate constants for each step and the difference extinction coefficient at 398 nm of HUPIII minus each intermediate species have been determined. The spectral characteristics of the intermediate species in the Soret region have been obtained buy rapid wavelength scanning stopped flow optical spctroscopy. The possible nature of the intermediate species are discussed in relation to data in Fig. 1. The amino acid sequence of HUPIII. The numbering of the residues refers to native cytochrome c (from ref. 11). the literature on the reactions of aquo Fe(III) myoglobin, catalase and horseradish peroxidase with H2O2.

The ficin-catalysed hydrolysis of p-nitrophenyl hippurate. Detailed kinetics including the measurement of the apparent dissociation constant for the enzyme-substrate complex.

It seems likely that high-resolution kinetic studies, involving the determination of the various intermediates in enzyme-catalysed reactions and their rates of interconversion under different experimental conditions, will eventually provide the necessary background for an understanding of structure-function relationships in catalytic proteins. In the present communication the basis of such a study is presented for the ficin-catalysed hydrolysis of p-nitrophenylhippurate. Several authors have presented evidence [l-4] that ficin and papain, like o-chymotrypsin [5,6], catalyze the hydrolysis of low-molecular weight substrates via a mechanism involving a minimum of two intermediates eq. (1)

A competition time-course method for following enzymic reactions applied to the hydrolysis of acetamide catalysed by an aliphatic amidase

The inducible amidase (acylamide amidohydrolase EC 3.5.1.4) from Pseudomonas aeruginosa catalyses a number of reactions, including the hydrolysis of shortchain amides such as acetamide and propionamide [ 11; the acyl transfer from acetamide to hydroxylamine and, at a far slower rate, the hydrolysis of ethyl acetate and other short-chain esters 121. At pH 7 these reactions can be represented by eq. (l)-(3):

The kinetics of the reaction of aquo Fe(III) myoglobin with hydrogen peroxide at pH 8

The kinetics of the reaction of aquo Fe(III) myoglobin (Mb(III)) with H2O2 at pH 8.0 and 20° have been investigated quantitatively under non-linear second order conditions. The data obtained at five concentrations of H2O2 (molar ratio [H2O2]/[Mb]total in the range 0.3 to 4) were analysed simultaneously by means of non-linear numerical integration and optimization techniques. The only mechanism that satisfied the triple requirement of a standard deviation within the standard error of the experimental data, good determination of the optimized parameters and a random distribution of residuals is as follows: Mb(III) reacts with H2O2 to form species X with a second order rate constant k+1 = 3.53 × 102 M−1 s−1; species X in turn reacts with H2O2 to form species R with a second order rate constant k+2 = 3.40 × 103 M-1s−1; species R is then converted back into Mb(III) with a first order rate constant k=3 = 9.60 × 10−5 s−1. At 408 nm ΔϵMb(III)−R = 71.9 mM−1 cm−1 and δϵMb(III)−X = 104 mM−1 cm−1. Species R is spectrally identical to the Fe(IV) myoglobin species described by George and Irvine [Biochem. J., 52, 511–517 (1952)]. The chemical nature of species X and R is discussed on the basis of the data in the present paper and data in the literature from EPR, Mossbauer, resonance Raman and magnetic susceptibility studies.