How important are entropic contributions to enzyme catalysis?

Abstract

The idea that enzymes accelerate their reactions by entropic effects has played a major role in many prominent proposals about the origin of enzyme catalysis. This idea implies that the binding to an enzyme active site freezes the motion of the reacting fragments and eliminates their entropic contributions, (⌬S cat ‡ )Ј, to the activation energy. It is also implied that the binding entropy is equal to the activation entropy, (⌬S w ‡ )Ј, of the corresponding solution reaction. It is, however, difficult to examine this idea by experimental approaches. The present paper defines the entropic proposal in a rigorous way and develops a computer simulation approach that determines (⌬S ‡ )Ј. This approach allows us to evaluate the differences between (⌬S ‡ )Ј of an enzymatic reaction and of the corresponding reference reaction in solution. Our approach is used in a study of the entropic contribution to the catalytic reaction of subtilisin. It is found that this contribution is much smaller than previously thought. This result is due to the following: (i ) Many of the motions that are free in the reactants state of the reference solution reaction are also free at the transition state. Many prominent proposals (e.g., see refs. 1 and 2) and textbooks that consider biochemical systems (e.g., refs. 3 and 4) invoke entropic contributions as major factors in enzyme catalysis. These proposals, which are intuitively very appealing (e.g., see ref. 5), have assumed that the large configurational space available for the reacting fragments in water would be drastically restricted in the enzyme active site. It has been thus deduced that this should lead to large entropic contributions to the difference between the activation barrier in the enzyme and in the reference solution reaction. However, the validity of these proposals is far from being obvious (6, 7). For example, the very inf luential proposal introduced by Page and Jencks (1) ref lects the assumption that the formation of the transition state in a bimolecular reaction in solution involves complete loss of three translational and three rotational degrees of freedom. However, two or more of these degrees of freedom are usually almost free in the transition state (see below). More serious is the implicit assumption that the entropic contribution to catalysis is given approximately by the negative of the binding entropy (see below). Other problems with simple estimates of the entropic contribution will be mentioned in the next section.The main stumbling block for determining the validity of the entropic proposal is the absence of direct experimental information about the corresponding contribution of the reacting fragments to the activation entropy in the enzyme and in solution. In this respect it is interesting to note the recent analysis of cytidine deaminase by Wolfenden and co-workers (8). This study found that the entropies of activation in the enzyme and in water are very similar and that the overall catalysis is due to enthalpic effects. Interestingly, it was found that ...

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