Contribution of buried distal amino acid residues in horse liver alcohol dehydrogenase to structure and catalysis

Abstract

The dynamics of enzyme catalysis range from the slow time scale (∼ms) for substrate binding and conformational changes to the fast time (∼ps) scale for reorganization of substrates in the chemical step. The contribution of global dynamics to catalysis by alcohol dehydrogenase was tested by substituting five different, conserved amino acid residues that are distal from the active site and located in the hinge region for the conformational change or in hydrophobic clusters. X-ray crystallography shows that the structures for the G173A, V197I, I220 (V, L, or F), V222I, and F322L enzymes complexed with NAD and an analogue of benzyl alcohol are almost identical, except for small perturbations at the sites of substitution. The enzymes have very similar kinetic constants for the oxidation of benzyl alcohol and reduction of benzaldehyde as compared to the wild-type enzyme, and the rates of conformational changes are not altered. Less conservative substitutions of these amino acid residues, such as G173(V, E, K, or R), V197(G, S, or T), I220(G, S, T, or N), and V222(G, S, or T) produced unstable or poorly expressed proteins, indicating that the residues are critical for global stability. The enzyme scaffold accommodates conservative substitutions of distal residues, and there is no evidence that fast, global dynamics significantly affect the rate constants for hydride transfers. In contrast, other studies show that proximal residues significantly participate in catalysis.

Substitutions of Amino Acid Residues in the Substrate Binding Site of Horse Liver Alcohol Dehydrogenase Have Small Effects on the Structures but Significantly Affect Catalysis of Hydrogen Transfer

“…relatively slow conformational changes, and relevant structures must be evaluated before effects of fast global dynamics on the transition state are invoked. 83,84 Challenges for Enzymology of Alcohol Dehydrogenases. The substitutions in the substrate binding site made only small changes in the structures of the enzyme− NAD + −pentafluorobenzyl alcohol complexes, apparent donor−acceptor distances, and temperature factors (Table 8), but the large changes in the enzyme kinetics (Table 5) suggest that motions in the active site (local dynamics) and energetics have been altered.…”

“…113,114 Of the residues identified, Val-203, Val-207, Glu-267, Ile-269, and Val-292 have been i n d i v i d u a l l y s u b s t i t u t e d a n d s t u d i e d i n h o r s e ADH. 9,18,22,78,84,115,116 In addition, five other distal, buried residues in the conserved motif, Gly-173, Val-197, Ile-220, Val-222, and Phe-322, were substituted and studied. 84 A M303F/ L308I/W314L triple substitution buried in the dimer interface has also been studied.…”

“…9,18,22,78,84,115,116 In addition, five other distal, buried residues in the conserved motif, Gly-173, Val-197, Ile-220, Val-222, and Phe-322, were substituted and studied. 84 A M303F/ L308I/W314L triple substitution buried in the dimer interface has also been studied. 117 The results showed that conservative substitutions of the residues that are not in the active site have modest effects on activity and structure, and no evidence is found to support a significant role for fast, global dynamics in catalysis.…”

“…119−121 The global conformational change is important for establishing the most active scaffold, but substitutions in the hinge region between domains (G173A and F322L) had only small effects on structure and catalysis. 84 Nevertheless, local motions can participate in the dance of substrates that leads to the "near-attack conformations" or tunneling ready states. 120 Among the ensemble of available conformational states, the catalytic courtship culminates in hydrogen transfer when the positions are favorable.…”

“…(The substitutions of Val-203 with Leu, Ala, or Gly in horse liver alcohol dehydrogenase also diminished hydrogen tunneling, but only the structure for the V203A enzyme has been determined − However, distal substitutions (of residues not in contact with substrates) can affect structures of active sites and catalysis by altering relatively slow conformational changes, and relevant structures must be evaluated before effects of fast global dynamics on the transition state are invoked. , …”