Thermodynamic nonideality in enzyme catalysis

Abstract

The enhanced catalytic reduction of pyruvate by rabbit muscle lactate dehydrogenase that results from the addition of serum albumin [Nichol, L. W., Sculley, M. J., Ward, L. D. & Winzor, D. J. (1983) Arch. Biochern. Biophys. 222,574 -5811 is shown to emanate solely from an increase in maximal velocity, there being no discernible effect of this inert space-filling macromolecular solute on the Michaelis constant for either pyruvate or NADII. As part of the search for a mechanistic explanation of this kinetic phenomenon, the space-filling effects of albumin have been used to eliminate the possibility that the increase in sedimentation coefficient of lactate dehydrogenase effected by inclusion of oxamate with enzyme-NADH complex reflects preferential binding of this pyruvate analog to a more compact isomeric state of the binary complex. 'The enzyme kinetic results are therefore considered in terms of a reaction scheme entailing gross conformational changes during the formation of ternary enzyme-NADH-pyruvate complex and its isomerization to an activated transition state. The experimentally observed insensitivity of the Michaelis constant for pyruvatc to albumin concentration is in keeping with theoretical prediction, but incorporation of the measured extent of maximal velocity enhancement into the kinetic model leads to a predicted volume for the fully saturated transition-state complex that is far too small to be experimentallyfeasible. A more complex mechanistic model involving additional isomerizations of enzyme-substrate species is thus required to achieve quantitative description of the albumin effect solely in terms of thermodynamic nonideality.The possible consequences of thermodynamic nonideality arising from the space-filling effects of inert solutes on enzyme systems were brought into prominence by the theoretical demonstration [I], in response to an experimental observation [2], that inclusion of solutes such as poly(ethy1ene glycol) could displace a self-association equilibrium from a point of nondetectability of polymer to a situation in which polymer is essentially the sole species present. Qualitatively similar effecls should also occur with enzymes undergoing substrate-induced conformational changes of sufficient magnitude to result in altered molecular dimensions (size/asymmetry) of the enzymc [3]. The fact that such displacement of an isomeric equilibrium is likely to be less extensive because of much smaller differences between the effective radii of isomers should not deter the search for the phenomenon, which is reported to remain detectable by enzyme kinetic studies [4 -61.The experimental observation that high concentrations of bovine serum albumin, ovalbumin and Dextran T70 all enhance the maximal velocity of pyruvate reduction by rabbit muscle lactate dehydrogenase [4] was considered unlikely to be the result of specific activation of the enzyme, and was therefore attributed to thermodynamic nonideality arising from the space-filling effects of inert macromolecular solutes on a substrate-ind...

Thermodynamic nonideality in macromolecular solutions

“…Several points should be noted. a) Although the results conform adequately with the theoretical requirement of a linear relationship between cib2 and rA (a correlation coefficient of 0.98), they are clearly not described by the broken line, which is the theoretical dependence that has been assumed in earlier investigations on the basis that Vp = 0.60 ml/g and rf = 0.3 nm for dextran [41, 571. b) Closer examination of Eqns (10)(11)(12)(13) of Edmond and Ogston [15] shows that conversion of the molar covolume (UAp) to the excluded volume/unit mass of polymer (cAp) entailed the substitution M p = nrf21fN/ip (10) where If is the length of the long rod (cylinder). Since the numerator on the right-hand side of Eqn (10) refers to the volume of the rod in aqueous solution, ip must take into account any hydration of the polymer for Mp to be the anhydrous molar mass used in conversions between molar and mass concentration scales.…”

Bilirubin Toxicity and Differentiation of Cultured Astrocytes

“…In addition, our experiments using lower concentrations showed significant toxicity of bilirubin in The presence of serum albumin has been shown to influence LDH quantification. 23,24 In our experience, HSA in the medium led to elevated measurement of LDH. Correction for this, however, did not interfere with the interpretation of our results.…”

A potential role for isothermal calorimetry in studies of the effects of thermodynamic non‐ideality in enzyme‐catalyzed reactions

“…An enhanced maximal velocity is thus the predicted consequence of molecular crowding in instances where the formation of ES z is accompanied by a decrease in volume ðB ES 0 ;M > B ES z ;M Þ-a phenomenon observed in kinetic studies with rabbit muscle lactate dehydrogenase (Nichol et al, 1983;Bergman and Winzor, 1989b). Conversely, a diminished maximal velocity in a concentrated molecular environment is predicted for systems in which the activated enzyme-substrate complex is the larger species ðB ES 0 ;M < B ES z ;M Þ-a situation encountered in the adenosine deaminase reaction (Lonhienne and Winzor, 2001) as well as in thrombin catalysis (Lonhienne et al, 2003a).…”

“…Studies of the effect of cosolute inclusion on the molecular size of the enzyme are therefore usually used to afford a more definitive means of deciding whether the equilibrium being affected by molecular crowding involves self-association or isomerization of the enzyme (Bergman and Winzor, 1989b;Chebotareva et al, 2001;Winzor, 2001, 2002;Lonhienne et al, 2003a).…”