So Do We Understand How Enzymes Work?

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So do we understand how enzymes work?
David Blow
Figure 1 (a) A B (b) A B (c)

Between 1930 and 1975 biochemical and structural analysis of enzymes led to a clear set of ideas that might form a basis for detailed understanding of enzyme action. Further development required energetic and thermodynamic analysis of enzymes in an aqueous medium, beyond the computational powerthen available. Structural enzymology advanced in other directions, but the fundamental questions of enzyme action must soon be re-opened.
Address: Blackett Laboratory, Imperial College, London SW7 2BZ, UK. E-mail: d.blow@ic.ac.uk Based on a keynote lecture given at the Howard Hughes Medical Institute, Bethesda, Maryland, USA on 7 November 1999. Structure 2000, 8:R77–R81 0969-2126/00/$ – seefront matter © 2000 Elsevier Science Ltd. All rights reserved.

A

B

(d) A OH H B

(e) OH A H B

(f) OH A H B

Structure

Our understanding of enzymes has developed incredibly during my lifetime. Friedrich Hunefeld crystallised haemoglobin 160 years ago. But when James Sumner crystallised jack bean urease in 1926, the biochemical establishment refused to believe that the crystalsrepresented the enzyme. The 1929 edition of the Encyclopaedia Britannica, recognised as a classic edition for the erudition of its contributors and the accuracy of its articles, states in the article on enzymes ‘The chemical structure of enzymes is not yet known. They were formerly regarded as proteins... [but are now conceived as]... a colloidal carrier and a purely chemical active group’. This errormanifests the extraordinary catalytic power of enzymes. Purified preparations containing no detectable protein (by methods then available) possessed obvious enzymatic activity. Richard Willstätter, doyen of enzyme chemists in the 1920’s, concluded that the protein (‘colloidal’) component was a non-specific carrier of adsorbed chemical catalytic agents which were the true enzymes [1]. Thecrystallisation of several hydrolytic enzymes by John Northrop and Moses Kunitz in the early years of my life resolved the question beyond reasonable doubt. Enzymes are proteins. In those same years, Desmond Bernal, with Dorothy Crowfoot and Max Perutz, found they could get good X-ray diffraction patterns from protein crystals, including chymotrypsin and pepsin, if they kept them moist. The unit cells werehuge. JBS Haldane’s book ‘Enzymes’ (1930) [2] maintains a careful neutrality about the chemical nature of enzymes,

Schematic representation of the mechanism of a proteolytic enzyme based on Haldane’s diagrams [2]. Parts (a), (b), (e) and (f) closely follow Haldane’s original. Parts (c) and (d) suggest how strain might affect the substrate in intermediate stages. Colours are used to indicatephysical stress on the enzyme — yellow and red for tension, blue and purple for compression.

but presents thought-provoking insight into their mode of action. An enzyme might catalyse a hydrolytic reaction if the substrate components ‘when combined with the enzyme, lay slightly further apart than their equilibrium distance when combined... but nearer than their equilibrium distance when free....Using Fischer’s lock and key simile, the key does not fit the lock perfectly but exercises a certain strain on it.’ Figure 1 is a slightly developed version of Haldane’s stimulating diagram. Alfred Mirsky and Linus Pauling [3] described proteins with startling modernity in 1936. A native protein molecule, they said, ‘consists of one... [or more] polypeptide chains which continue withoutinterruption throughout the molecule... folded into a uniquely defined configuration... held together by hydrogen bonds between the peptide nitrogen and oxygen atoms and also between [charged sidechains]... The denatured protein molecule we consider to be characterized by the absence of a uniquely defined conformation.’ Despite the accuracy of this insight, the question as to whether a protein was a...