Gösta Pettersson

Liver alcohol dehydrogenase.

The article deals with the structure and function of liver alcohol dehydrogenase and reviews mainly literature published after 1979, i.e., summarizes progress made in the field since Klinman presented her review on alcohol dehydrogenases. The emphasis will be on high-resolution crystallographic data, results obtained with metal-substituted enzyme derivatives, and on the mechanism and pH dependence of the catalytic reaction.

Kinetics of the coupled reaction catalysed by a fusion protein of β-galactosidase and galactose dehydrogenase

The mechanistic implications of the kinetic behaviour of a fusion protein of beta-galactosidase and galactose dehydrogenase have been analysed in view of predictions based on experimentally determined kinetic parameter values for the galactosidase and dehydrogenase activities of the protein. The results show that the time course of galactonolactone formation from lactose in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is given to the mutarotation of the reaction intermediate galactose. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses galactonolactone formation from lactose by a mechanism involving channelling of galactose.

Soaking in Cs2SO4 reveals a caesium-aromatic interaction in bovine-liver rhodanese

Soaking crystals of rhodanese (thiosulphate:cyanide sulphurtransferase) in 2 M caesium sulphate reveals three caesium binding sites of this enzyme. One of these had been described before as a binding site for sodium ions and is located in a cleft close to the active site. In this site the monovalent cation is coordinated by five oxygen atoms. The first additional binding site seems to be quite special. The caesium ion is bound to the phenyl ring of a tryptophan residue. It is further liganded by two oxygen atoms. The third binding site is a result of crystal packing effects: caesium is liganded by four oxygen atoms, provided by two rhodanese molecules and one sulphate ion. It is likely that the binding of caesium affects the fluorescence of the tryptophan residue with which it interacts. Such possible effects should also be kept in mind when caesium ions are used as a quencher in fluorescence studies of proteins in general.

Substrate-inhibition by acetyl-CoA in the condensation reaction between oxaloacetate and acetyl-CoA catalyzed by citrate synthase from pig heart

Deviations from Michealis-Menten kinetics in the pig-heart citrate synthase (citrate-oxaloacetate-lyase(pro-3S-CH2-COO-leads to acetyl-CoA), EC 4.1.3.7) system have been characterized and analyzed in view of the kinetic theory described in the preceding paper. The enzymic condensation reaction between acetyl-CoA and oxaloacetate is subject to substrate-inhibition by acetyl-CoA. This can be attributed to the formation of a productive enzyme-acetyl-CoA complex with a dissociation constant of 110 uM. The binding of acetyl-CoA to the enzyme decreases the on-velocity constant for oxaloacetate-binding from 4000 min-1- micrometer-1 to 1700 min-1-micrometer-1. The affinity of citrate synthase for oxaloacetate increase at least 20-fold on the binding of acetyl-CoA. The latter cooperativity effect can be attributed to a more than 45-fold decrease of the off-velocity constant for oxaloacetate-binding.

Dalziel rate behaviour in ternary-complex mechanisms for enzyme reactions involving two substrates

Rapid-equilibrium and compulsory-order cases are not exclusive in giving rise to a reciprocally bilinear initial rate behaviour in the generalized ternary-complex mechanism for enzymatic two-substrate reactions shown in Scheme 1. General expressions relating empirical Dalziel coefficients to velocity and equilibrium constants in Scheme 1 are derived and show that rapid-equilibrium and compulsory-order equations may be considered as opposite extremes of a general empirical Dalziel rate equation for the random-order ternary-complex mechanism. Kinetic characteristics of non-equilibrium ternary-complex mechanisms are described and discussed in view of the special case for which the second-degree rate equation corresponding to Scheme 1 becomes mathematically identical with a bilinear Dalziel equation. Kinetic differences between the bilinear special cases inherent in Scheme 1 primarily concern the Dalziel coefficients ϕ1 and ϕ2. Empirical Dalziel coefficients obtained for enzymes operating by this mechanism should always be interpreted in view of the general Dalziel equation, and any reduction of this relationship to those previously derived for rapid-equilibrium and compulsory-order cases must be supported by experimental evidence. General criteria imposing previously not recognized restrictions on the quotients ϕ1/ϕ12 and ϕ2/ϕ12 in comparison to equilibrium constants for the

Optimal kinetic design of enzymes in a linear metabolic pathway

The rate equations for a sequence of enzymic reactions conforming to Michaelis-Menten kinetics have been analyzed in order to establish what kinetic design optimizes the steady-state reaction flux for a given total concentration of enzymes and a given average magnitude of true and apparent first-order rate constants in the reaction system. Analytical solutions are presented which have been derived with the assumptions that the concentration of the first substrate in the pathway represents a fixed parameter and that no diffusional constraints come into operation. The solutions prescribe that reaction flux in the examined system becomes optimal when all of the enzymes are present at equal active-site concentrations. The optimal kinetic design of each enzyme reaction is characterized by forward (true or apparent) first-order rate constants of equal magnitude and reverse rate constants of equal magnitude. This means that the optimal kinetic design of the examined pathway is highly uniform, individual enzymes being likely to exhibit optimal V values differing by a factor less than 5 and optimal Km/[S] values falling within the range 0.3-2.

Control properties of the Calvin photosynthesis cycle at physiological carbon dioxide concentrations

A previously described kinetic model for the Calvin cycle and ancillary pathway of starch production in the chloroplast of C3 plants has been extended so that it becomes applicable under physiological conditions where there is a competition between carbon dioxide and oxygen for ribulose-1,5-bisphosphate carboxylase (rubisco). The modified model is shown to account for the observed dependencies of the rate of carbon dioxide assimilation in leaves on the concentrations of carbon dioxide, oxygen, and rubisco. The predictions of the model are examined with particular regard to the control characteristics of the photosynthetic process of carbohydrate formation under physiological conditions.

Error associated with experimental flux control coefficient determinations in the Calvin cycle

Model studies of photosynthetic carbohydrate formation in the chloroplast of C3 plants have been performed to examine the expected response of the steady-state reaction flux to finite changes in concentration or activity of individual enzymes in a central metabolic network. The results indicate that flux control coefficients in this system cannot be reliably estimated experimentally even for the two enzymes that do exert predominant control under the examined conditions. Reduction of the mathematical error of the estimates to a satisfactorily low level requires such small enzyme concentration changes that presently available assay methods do not allow for a determination of the corresponding flux changes with satisfactory precision. It is concluded from these observations and general methodological considerations that experimental flux control coefficient estimates cannot be trusted unless evidence is presented to show that the mathematical error associated with their determination is of insignificant magnitude.

Substrate-inhibition and substrate-activation in the random-order ternary-complex mechanism for enzyme reactions involving two substrates

Abstract Deviations from Michaelis-Menten kinetics in the random-order ternary-complex mechanism for enzyme reactions involving two substrates (Scheme 1) have been analyzed in view of the asymptote theory for higher-degree rate equations. The patterns of substrate-inhibition or substrate-activation inherent in this mechanism are characterized. Generalized relatinships for the evaluations and interpretation of such kinetic patterns in terms of rate constants in the mechanism are derived, and the appropriate reduction of these relationships in some special cases of particular interest is described and discussed.

Mechanism of NADH transfer among dehydrogenases

Steady-state and transient-state kinetic experiments have been performed to test the proposal that there is a direct (channelled) transfer of NADH from one dehydrogenase to another if the two enzymes exhibit distinct chiral coenzyme specificity (A-side vs. B-side). The results lend no support to this proposal, but are fully consistent with a free-diffusion mechanism of NADH transfer irrespective of the chiral specificity of the enzymes.