“…Ac ombination of steady-state kinetics, [4,9,11] isotope effects, [15] site-directed mutagenesis [4] and crystallographic studies, [5a-c,e, 6] mainly on E. coli ands pinachK ARI, has led to the proposal that both the isomerisation and reduction reactions requiret wo metal ions and NADPH, and these need to be present in the same active site, since the isomerisation reaction alone is thermodynamically unfavourable (i.e.,t he equilibrium of the reaction is strongly on the side of the substrate). Indeed,n oi ntermediate of the reactioni sb eing released, despite the observation that the product of the isomerization reaction (i.e.,3 -hydroxy-3-methyl/ethyl-2-ketobutyrate) is an excellent substrate, but with relatively modest binding affinity.…”