Heavy Enzymes and the Rational Redesign of Protein Catalysts

Abstract

An unsolved mystery in biology concerns the link between enzyme catalysis and protein motions. Comparison between isotopically labelled "heavy" dihydrofolate reductases and their natural-abundance counterparts has suggested that the coupling of protein motions to the chemistry of the catalysed reaction is minimised in the case of hydride transfer. In alcohol dehydrogenases, unnatural, bulky substrates that induce additional electrostatic rearrangements of the active site enhance coupled motions. This finding could provide a new route to engineering enzymes with altered substrate specificity, because amino acid residues responsible for dynamic coupling with a given substrate present as hotspots for mutagenesis. Detailed understanding of the biophysics of enzyme catalysis based on insights gained from analysis of "heavy" enzymes might eventually allow routine engineering of enzymes to catalyse reactions of choice.

Keywords: alcohol dehydrogenases; dihydrofolate reductases; enzyme engineering; isotope effects; molecular dynamics.

Figure 1

Strategies for the production and analysis of heavy enzymes. A) to D) show A) the production of natural‐abundance enzyme, B) whole‐enzyme isotope labelling, C) single‐residue isotope labelling, and D) segmental isotope labelling by production of two peptides, only one of which is labelled with heavy isotopes, that are ligated together and refolded. E) How heavy enzymes are analysed by kinetics and computational analysis.

Scheme 1

Reaction catalysed by dihydrofolate reductase.

Figure 2

Temperature dependent pre‐steady‐state KIEs for different DHFRs at pH 7.0. Data show MpDHFR (•),12i EcDHFR (⧫)12j and BsDHFR (▴).12h The arrows indicate the temperature at which the KIE tends to unity for psychrophilic MpDHFR, mesophilic EcDHFR and thermophilic BsDHFR.

Scheme 2

A) Reaction catalysed by BsADH. B) A biocatalytic application of BsADH to generate a valuable compound.23

Figure 3

BsADH heavy enzyme KIE for a range of substrates correlated with k _cat at 20 °C. A) Structures of substrates tested. B) Enzyme KIEs are shown in circles; k _cat values are shown as bars. Data from ref. 12k.