Catalytic efficiency and kcat/KM: a useful comparator?

doi:10.1016/j.tibtech.2007.03.010

Trends in Biotechnology

Volume 25, Issue 6, June 2007, Pages 247-249

https://doi.org/10.1016/j.tibtech.2007.03.010 Get rights and content

The ratio k_cat/K_M – often referred to as the ‘specificity constant’ – is a useful index for comparing the relative rates of an enzyme acting on alternative, competing substrates. However, an alternative description, ‘catalytic efficiency’, is frequently used, and on occasions misused, to compare the reactivity of two enzymes acting on the same substrate. Here, we highlight the pitfalls in using k_cat/K_M to compare the catalytic effectiveness of enzymes.

Introduction

The power provided by recombinant DNA techniques (e.g. site-directed mutagenesis and directed evolution) has enabled the production of enzymes ‘engineered’ to fit the requirements of technological applications. When considering the catalytic rate exhibited by a library of enzymes catalyzing the same reaction, the question arises as to which is the ‘better’ enzyme (often expressed by terms such as catalytic proficiency), and whether it is possible to describe relative catalytic effectiveness in quantitative terms. Such considerations also concern aspects of enzyme evolution, in the Darwinian sense.

Accepting that enzymes operate mostly under steady-sate conditions, both in vivo and in vitro, the relevant kinetic parameters of an enzyme that are determined are k_cat (the catalytic constant for the conversion of substrate to product) and K_M (the Michaelis constant, which is defined, operationally, as the substrate concentration at which the initial rate is one-half of the maximum velocity). In fact, it is the ratio of these two parameters, k_cat/K_M, that is often used when comparing enzymes. In this article we explore this approach and suggest that, in general, when comparing enzymes catalyzing the same reaction, the use of k_cat/K_M as a quantitative index of catalytic power is at best misleading and at worst invalid.

Section snippets

One enzyme, two substrates

The term k_cat/K_M is often used as a specificity constant to compare the relative rates of reaction of each of a pair of substrates, when each is catalytically transformed by an enzyme. This is because, if K_M is used on its own as the indicator of specificity, the effect of the ‘better’ substrate will be strongly manifested mainly at values of [S]/K_M << 1. As [S]/K_M increases above this value, k_cat becomes the parameter that best describes the better substrate. This dichotomy is resolved by using k

Comparing two enzymes

However, throughout the past two decades a regrettable extension of the use of k_cat/K_M has been the increasingly prevalent use of this ratio as an index for comparing different enzymes. This is particularly commonplace in reports dealing with enzymes that are mutated (or engineered) with a view to altering the steady-state kinetic parameters to produce more powerful catalysts. When used in this context, k_cat/K_M is variously called ‘catalytic efficiency’, ‘catalytic potential’, ‘performance

Recommended use of k_cat/K_M

We have demonstrated that in a general case an enzyme having a higher catalytic efficiency (i.e. k_cat/K_M value) can, at certain substrate concentrations, actually catalyze an identical reaction at lower rates than one having a lower catalytic efficiency. Even where the enzyme with the higher k_cat/K_M catalyzes a reaction faster than one with a lower k_cat/K_M, the ratio of the two reaction rates is not a constant, but depends on the value of [S]/K_M (except in the special case where the K_M values

Acknowledgements

We thank Athel Cornish-Bowden and Nick Price for helpful advice and discussions. Work in the authors’ laboratory is supported by the Royal Society and the Biotechnology and Biological Sciences Research Council (UK).

References (3)

D.E. Koshland
The application and usefulness of the ratio k_cat/K_M
Bioorg. Chem.
(2002)

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Trends in Biotechnology

OpinionCatalytic efficiency and kcat/KM: a useful comparator?

Introduction

Section snippets

One enzyme, two substrates

Comparing two enzymes

Recommended use of kcat/KM

Acknowledgements

Bioorg. Chem.

Opinion
Catalytic efficiency and k_cat/K_M: a useful comparator?

Recommended use of k_cat/K_M