The Activation Parameters of a Cold-Adapted Short Chain Dehydrogenase Are Insensitive to Enzyme Oligomerization

Abstract

The structural principles of enzyme cold adaptation are of fundamental interest both for understanding protein evolution and for biotechnological applications. It has become clear in recent years that structural flexibility plays a major role in tuning enzyme activity at low temperatures, which is reflected by characteristic changes in the thermodynamic activation parameters for psychrophilic enzymes, compared to those of mesophilic and thermophilic ones. Hence, increased flexibility of the enzyme surface has been shown to lead to a lower enthalpy and a more negative entropy of activation, which leads to higher activity in the cold. This immediately raises the question of how enzyme oligomerization affects the temperature dependence of catalysis. Here, we address this issue by computer simulations of the catalytic reaction of a cold-adapted bacterial short chain dehydrogenase in different oligomeric states. Reaction free energy profiles are calculated at different temperatures for the tetrameric, dimeric, and monomeric states of the enzyme, and activation parameters are obtained from the corresponding computational Arrhenius plots. The results show that the activation free energy, enthalpy, and entropy are remarkably insensitive to the oligomeric state, leading to the conclusion that assembly of the subunit interfaces does not compromise cold adaptation, even though the mobilities of interfacial residues are indeed affected.

Reply to Comment on: “Computer Simulations Reveal an Entirely Entropic Activation Barrier for the Chemical Step in a Designer Enzyme”

“…Hence, all of the loops covering the active site are fully mobile and it is, in fact, only 163 atoms (belonging to 16 residues) on the back side of the enzyme that are restrained. This type of simulation model has been shown earlier for numerous systems to give accurate values of thermodynamic activation parameters compared to experiment. − In particular, the model has also been shown to capture the curved Arrhenius plot for a cold-adapted α-amylase, demonstrating that the curvature is not due to a heat capacity effect but to the existence of an inactive state of the ES complex . This has recently also been verified by us experimentally by redesign of the α-amylase .…”

Efficient Empirical Valence Bond Simulations with GROMACS

“…To demonstrate the validity of our EVB protocol, we chose to replicate two computational experiments that have already been published by our group in refs and . From ref , we simulated the conversion between 3-oxovalerate and ( R )-3-hydroxyvalerate catalyzed by the psychrophilic ( R )-3-hydroxybutyrate dehydrogenase from Psychrobacter arcticus ( Pa HBDB) in its monomeric form. This is a concerted reaction where a hydride ion is transferred from the NADH cofactor to the substrate simultaneously with proton transfer from Tyr161 (Figure ).…”

“…To check the validity of our protocol, we chose to replicate two of our previous computational experiments which have already been published in refs and . The first of these is the NADH-dependent reduction of 3-oxovalerate to ( R )-3-hydroxyvalerate catalyzed by the psychrophilic ( R )-3-hydroxybutyrate dehydrogenase .…”

“…To check the validity of our protocol, we chose to replicate two of our previous computational experiments which have already been published in refs and . The first of these is the NADH-dependent reduction of 3-oxovalerate to ( R )-3-hydroxyvalerate catalyzed by the psychrophilic ( R )-3-hydroxybutyrate dehydrogenase . The second case is the first proton transfer step in the conversion between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) catalyzed by yeast triosephosphate isomerase (TIM) …”

“…The first of these is the NADH-dependent reduction of 3-oxovalerate to (R)-3hydroxyvalerate catalyzed by the psychrophilic (R)-3-hydroxybutyrate dehydrogenase. 12 The second case is the first proton transfer step in the conversion between dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (GAP) catalyzed by yeast triosephosphate isomerase (TIM). 13 ■ METHODS EVB Model.…”