Enzyme dynamics and hydrogen tunnelling in a thermophilic alcohol dehydrogenase

Abstract

Biological catalysts (enzymes) speed up reactions by many orders of magnitude using fundamental physical processes to increase chemical reactivity. Hydrogen tunnelling has increasingly been found to contribute to enzyme reactions at room temperature. Tunnelling is the phenomenon by which a particle transfers through a reaction barrier as a result of its wave-like property. In reactions involving small molecules, the relative importance of tunnelling increases as the temperature is reduced. We have now investigated whether hydrogen tunnelling occurs at elevated temperatures in a biological system that functions physiologically under such conditions. Using a thermophilic alcohol dehydrogenase (ADH), we find that hydrogen tunnelling makes a significant contribution at 65 degrees C; this is analogous to previous findings with mesophilic ADH at 25 degrees C. Contrary to predictions for tunnelling through a rigid barrier, the tunnelling with the thermophilic ADH decreases at and below room temperature. These findings provide experimental evidence for a role of thermally excited enzyme fluctuations in modulating enzyme-catalysed bond cleavage.

Isotope Substitution of Promiscuous Alcohol Dehydrogenase Reveals the Origin of Substrate Preference in the Transition State

“…Hence, substrate KIEs (KIE= k cat H / k cat D ) can be determined by measuring the steady‐state rate constant ( k cat ) using protiated and deuterated substrates. In agreement with previous work,6a, 7c the resulting substrate KIE is highly temperature‐dependent, increasing sharply at lower temperatures but remaining constant above 40 °C (Figure 2 A and Figures S1, S2 in the Supporting Information). The natural substrates of BsADH are generally small molecules, which serve as hydride acceptors under anaerobic conditions 9.…”

Linking protein structure and dynamics to catalysis: the role of hydrogen tunnelling

“…As observed initially in the reaction catalysed by soybean lipoxygenase (SLO; Glickman et al 1994;Hwang & Grissom 1994) and, subsequently, in a wide range of other enzyme-catalysed C-H abstraction reactions (e.g. Basran et al 1999Basran et al , 2001Kohen et al 1999;Abad et al 2000;Harris et al 2000;Francisco et al 2002;Sikorski et al 2004), the value of A 1 /A 2 often lies considerably above unity. In one case (dihydrofolate reductase, (Sikorski et al 2004)), a complex set of conditions have been postulated to explain A 1 /A 2 [1 within the tenets of variational transition state theory (VTST; Pu et al 2005).…”

“…The reactions catalysed by the alcohol dehydrogenases from yeast, horse liver and Bacillus stearothermophilus have provided some of the most direct evidence for aberrant Swain-Schaad relationships that lie outside these semi-classical limits (Cha et al 1989;Bahnson et al 1997;Kohen et al 1999;table 1). It is noteworthy that the summarized data have been observed for the secondary (non-transferred) hydrogen of alcohol substrates doubly labelled at the methylene position with either LZD, T or H, T; i.e.…”

“…The best-studied enzyme of this class with regard to isotope effect behaviour is the ht-ADH. This remarkable enzyme shares a common transition (at ca 30 8C) in both its tunnelling behaviour (Kohen et al 1999) and protein flexibility (as measured by hydrogen/deuterium exchange; Liang et al 2004a,b). The data support a model in which the enzyme undergoes a temperaturedependent transition that leads to a local increase in the flexibility of the protein.…”

“…and (iii) enzyme forms that have been mutated at their active sites (table 2; Cha et al 1989;Rucker et al 1992;Bahnson et al 1993Bahnson et al , 1997Kohen et al 1999). The latter involves either an increase in bulk at Leu57 or Phe93 (numbering refers to the horse liver alcohol dehydrogenase (LADH)), to alter the rate of substrate release from the active site pocket and make chemistry more rate determining, or a reduction in bulk at Val203 to alter its interactions with the C-4, reactive position of cofactor.…”