Three-Dimensional Views of Enzymes and How They Work

Three-Dimensional Views of Enzymes and How They Work 

In order to understand mechanisms of enzyme catalysis not only are the tertiary structures of enzymes of interest hut so too are the tertiary structures of enzyme-suhstrate complexes. There was, however, a problem As incredibly efficient catalysts, enzymes turn over substrate molecules in fractions of a second, while (40 years ago) collection of crystallographic data took days. The answer to the problem was to study complexes of exceedingly sluggish substrates, unreactive model substrates, as well as strongly bound inhibitors that compete for the active site. 

At Stanford, Harden M. McConnell developed a new technique, called spin labelling, based upon EPR spectroscopy. While carbon-centered free radicals are extremely reactive and short-lived, radical oxides of nitrogen, such as NO and NO2, are moderately stable. McConnell noted that nitroxyl radicals (RR N-O) are extremely stable if R and R are tertiary and can be chemically attached to biological molecules of interest. In 1965, he published the concept of spin labeling and, in 1966, demonstrated that a spin-labelled substrate added to a-chymotrypsin forms a covalent enzyme-substrate complex. The EPR signal was quite broad suggesting restricted motion consistent with Koshland s induced-fit model. In 1971, McConnell published a smdy in which spin labelling indicated flip-flop motions of lipids in cell membranes. This was the start of dynamic smdies of cell membranes. 

This first decade of crystallographic study of enzymes helped to create two new fields bio-inorganic chemistry and mechanistic bio-organic chemistry. Harry B. Gray (1935- ), at Caltech, began to make fundamental contributions to the chemistry and chemical physics of metal-loproteins. Unlike reactions between transition metal ions and their complexes, which involve intimate contact and electron-transfer upon collision, electron transfer between metals on different proteins is not so intimate and may require several collisions and may proceed through 

One of the most fascinating catalytic systems is the serine 195/histi-dine 57/aspartic acid 102 triad in the proteolytic enzyme chymotrypsin. It was discovered in 1969 by David M. Blow (1931-2004) at Cambridge. The presence of ionic groups in a hydrophobic reaction site enormously increases the basicities and reactivities of these catalytic groups. 

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