The catalytic power of enzymes: Conformational selection or transition state

stabilization?

Abstract

The mechanism by which enzymes produce enormous rate enhancements in the reactions

they catalyze remains unknown. Two viewpoints, selection of ground state conformations

and stabilization of the transition state, are present in the literature in apparent opposition. To

provide more insight into current discussion about enzyme efficiency, a two-state model

of enzyme catalysis was developed. The model was designed to include both the pre-

chemical (ground state conformations) and the chemical (transition state) components of the

process for the substrate both in water and in the enzyme. Although the model is of general

applicability, the chorismate to prephenate reaction catalyzed by chorismate mutase was

chosen for illustrative purposes. The resulting kinetic equations show that the catalytic power

of enzymes, quantified as the kcat/kuncat ratio, is the product of two terms: one including the

equilibrium constants for the substrate conformational states and the other including the rate

constants for the uncatalyzed and catalyzed chemical reactions. The model shows that these

components are not mutually exclusive and can be simultaneously present in an enzymic

system, being their relative contribution a property of the enzyme. The developed

mathematical expressions reveal that the conformational and reaction components of the

process perform differently for the translation of molecular efficiency (changes in energy

levels) into observed enzymic efficiency (changes in kcat), being, in general, more productive

the component involving the transition state.

Problem Statement

Enzymes are biological catalysts producing rate enhancements up to 1017 fold with respect to

uncatalyzed reactions in water. In spite of the vast amount of data in the literature, a complete

explanation concerning enzyme efficiency remains open. In particular, the question whether
the catalytic power of enzymes involves the stabilization of the transition state (TS) or the

selection of ground state (GS) conformations is under debate. In this regard, the proposal of

Pauling that an enzyme achieves catalysis only by net stabilization of the TS has been a

central paradigm in enzymology during years. However, recent computational studies on the

chorismate to prephenate reaction catalyzed by chorismate mutase (CM) suggested that the

rate of the reaction is strongly dependent on the formation of GS conformers that can convert

directly to the TS.

In this study, a kinetic model of enzyme catalysis which includes both the conformational

(pre-chemical) and the TS (chemical) components will be explored. Our aim was to help to

bridge the gap between these apparent opposite views. To this end, our approach focuses on

characterizing the translation of these molecular properties into meaningful kinetic

expressions to allow a quantitative analysis of their relative contribution to enzyme efficiency.

The CM was selected as an example as this enzyme is a key system for the current debate.

Nevertheless, the ideas and equations herein presented are intended to be of general

applicability.

General purpose

 Discover if the catalytic power of the enzymes comes from the transition state (TS) or

ground state (GS).

Theoretical framework

a) What are the enzymes?

Enzymes are proteins, polymers composed by chemically bonded aminoacid molecules,

which catalyze a wide range of chemicals reactions inside organisms. The catalytic activity of

enzymes depends on their three-dimensional (3D) structure. Inside these 3D structures there

are cavities, called “active site”, that shows affinity for specific molecules (called substrates)

that will eventually become products. The combination of chemical functional groups in the
active site creates covalent and non-covalent interactions between the protein and the

substrate molecules; these interactions favor the conversion of substrates into products. As

any catalyst, after the substrate transformation and once the product have been released from

the active site, the enzyme returns to a basal or ground state (GS) and is ready to engage in a

new catalytic cycle. Enzymes function outside cells, and historically men has used enzymes

for their benefit. The most ancient enzyme applications are related to food, such as the

production of bread and cheese. In this article we explain how efficient enzymes are and how

do they work. We also present a brief account on the history of their discovery and the

scientific breakthroughs that allowed the development of biocatalysts as one of the most

significant biotechnologies in the modern era.

b) Transition State (TS)

The transition state is the transitory of molecular structure in which the molecule is no longer

a substrate but not yet a product. All chemical reactions must go through the transition state

to form a product from a substrate molecule. The transition state is the state corresponding to

the highest energy along the reaction coordinate. It has more free energy in comparison to the

substrate or product; thus, it is the least stable state. The specific form of the transition state

depends on the mechanisms of the

particular reaction.

In the equation S → X → P, X is the

transition state, which is located at the peak

of the curve on the Gibbs free energy graph.

 c) Aplication to enzyme

Enzymes are usually proteins that act like catalysts. The enzyme's ability to make the reaction

faster depends on the fact that it stabilizes the transition state. The transition state's energy or,

in terms of a reaction, the activation energy is the minimum energy that is needed to break

certain bonds of the reactants so as to turn them into products. Enzymes decreases activation

energy by shaping its active site such that it fits the transition state even better than the

substrate. When the substrate binds, the enzyme may stretch or distort a key bond and

weaken it so that less activation energy is needed to break the bond at the start of the reaction.

In many cases, the transition state of a reaction has a different geometry at the key atom (for

instance, tetrahedral instead of trigonal planar). By optimizing binding of a tetrahedral atom,

the substrate is helped on its way to the transition state and therefore lowers the activation

energy, allowing more molecules to be able to turn into products in a given period of time.

The enzyme stabilizes the transition state through various ways. Some ways an enzyme

stabilizes is to have an environment that is the opposite charge of the transition state,

providing a different pathway, and making it easier for the reactants to be in the right

orientation for reaction.