TY - JOUR
T1 - Fast protein motions are coupled to enzyme H-transfer reactions
AU - Pudney, Christopher
AU - Guerriero, Andrew
AU - Baxter, Nicola Jane
AU - Johannissen, Linus O
AU - Waltho, Jonathan Peter
AU - Hay, Sam
AU - Scrutton, Nigel S
PY - 2013/2
Y1 - 2013/2
N2 - Coupling of fast protein dynamics to enzyme chemistry is controversial and has ignited considerable debate, especially over the last 15 years in relation to enzyme-catalysed H-transfer. H-transfer can occur by quantum tunneling and the tempera-ture-dependence of kinetic isotope effects (KIEs) has emerged as the ‘gold standard’ descriptor of these reactions. The anomalous temperature dependence of KIEs is often rationalised by invoking fast motions to facilitate H-transfer, yet crucially, direct evidence for coupled motions is lacking. Here, we have perturbed vibrational motions in pentaerythritol tetranitrate reductase (PETNR) by isotopic substitution where all non-exchangeable atoms were replaced with the corresponding heavy isotope (13C, 15N and 2H). The KIE temperature dependence is perturbed by heavy isotope labelling, demonstrating a direct link between (promoting) vibrations in the protein and the observed KIE. We show causality between fast motions and enzyme chemistry and demonstrate how this impacts on experimental KIEs for enzyme reactions
AB - Coupling of fast protein dynamics to enzyme chemistry is controversial and has ignited considerable debate, especially over the last 15 years in relation to enzyme-catalysed H-transfer. H-transfer can occur by quantum tunneling and the tempera-ture-dependence of kinetic isotope effects (KIEs) has emerged as the ‘gold standard’ descriptor of these reactions. The anomalous temperature dependence of KIEs is often rationalised by invoking fast motions to facilitate H-transfer, yet crucially, direct evidence for coupled motions is lacking. Here, we have perturbed vibrational motions in pentaerythritol tetranitrate reductase (PETNR) by isotopic substitution where all non-exchangeable atoms were replaced with the corresponding heavy isotope (13C, 15N and 2H). The KIE temperature dependence is perturbed by heavy isotope labelling, demonstrating a direct link between (promoting) vibrations in the protein and the observed KIE. We show causality between fast motions and enzyme chemistry and demonstrate how this impacts on experimental KIEs for enzyme reactions
UR - http://www.scopus.com/inward/record.url?scp=84874083777&partnerID=8YFLogxK
UR - http://dx.doi.org/10.1021/ja311277k
U2 - 10.1021/ja311277k
DO - 10.1021/ja311277k
M3 - Article
SN - 0002-7863
VL - 135
SP - 2512
EP - 2517
JO - Journal of the American Chemical Society
JF - Journal of the American Chemical Society
IS - 7
ER -