Effects of conformational selectivity and of overlapping kinetically influential ionizations on the characteristics of pH-dependent enzyme kinetics. Implications of free-enzyme pKa variability in reactions of papain for its catalytic mechanism

Abstract

The effects of selection by a small molecule, when binding to a protein, of a particular conformation from an equilibrium stereopopulation on the characteristics of the pH-dependence of reaction with a reactivity probe or substrate were determined by analysis of an appropriate kinetic model. For reaction in one protonic state containing an equilibrium mixture of two conformational isomers, the pH-second-order rate constant (k) profile is of conventional sigmoidal form. The apparent pKa value is a composite of the pKa values of the two conformational states. The value of pKapp. for a given enzyme under given experimental conditions will always be the same (provided that the site-specificity assumed in the model is maintained) irrespective of whether only one conformation reacts or both react, with the same or with different rate constants. The experimentally determined pH-independent rate constant (kapp.) is an average of the reactivities of the two conformational states weighted in favour of the predominant form. The presence of an additional but unreactive conformational state also affects the value of kapp. The possibility that overlapping acid dissociations that affect the reactivity of the enzyme might provide pH-k profiles often indistinguishable in practice from simple sigmoidal dissociation curves and subject to variability in apparent pKa values was evaluated by a simulation study. If two reactive protonic states of the enzyme respond differently to changes in the structure of the substrate or site-specific reactivity probe, differences in apparent pKa values of up to approx. 1 unit can be exhibited without deviation from sigmoidal behaviour being reliably observed. Differences in apparent pKa values observed in some site-specific reactions of papain and their possible consequences for its catalytic mechanism are discussed.

On the binding affinity of macromolecular interactions: daring to ask why proteins interact

“…A third mechanism of molecular recognition is the fluctuation (dynamic) fit [143] (figure 6 c ), also recently rediscovered and termed (among others) conformational selection [148,149], conformational selectivity [149], population shift [150], selected fit [151] and pre-existing equilibrium [152]. For consistency with current literature, the conformational selection term will be used here.…”

Identification of signalling and non-signalling binding contributions to enzyme reactivity. Alternative combinations of binding interactions provide for change in transition-state geometry in reactions of papain

“…9.5. The variability of observed pKa values characteristic of what is sometimes apparently a single ionization of the free enzyme molecule with change in probe or substrate structure resulting from overlapping ionizations has been explored previously (Brocklehurst et al, 1983).…”

From “fluctuation fit” to “conformational selection”: Evolution, rediscovery, and integration of a concept

“…Instead of a fit induced by the substrate, I would suggest a fluctuating enzyme molecule, one particular form of which is able to bind the substrate and other forms another ligand ” 10. Using the same method of assessing sulfhydryl reactivity, cysteine proteases have provided further experimental evidence for co‐existing molecular conformations in protein‐ligand mixtures 11, 12. H‐isotope exchange experiments 13, 14 maintained this concept until the advent of molecular dynamics simulations and high‐resolution proton NMR measurements on proteins.…”

“…Besides the terms “fluctuation fit” and “conformational selection”, several other less widely used phrases have also been introduced: “ conformational selectivity ” 12, “ stabilization of conformational ensembles ” 16, and more recently, “ population‐shift ” 39, “ selected fit ” 3, and “ pre‐existing equilibrium ” 40. The earlier works 12, 16 discussed the phenomenon of molecular recognition and the induced fit concept, in light of studies by Straub and co‐workers. The latter studies compared the two mechanisms from the perspective of kinetics 3, the energy landscape theory 39 or on the basis of elastic network models 40.…”