Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures

Biochemistry
S Tsai, J P Klinman

Abstract

The temperature dependence of steady-state kinetics has been studied with horse liver alcohol dehydrogenase (HLADH) using protonated and deuterated benzyl alcohol as substrates in methanol/water mixtures between +3 and -50 degrees C. Additionally, the competitive isotope effects, k(H)/k(T) and k(D)/k(T), were measured. The studies indicate increasing kinetic complexity for wild-type HLADH at subzero temperatures. Consistent with earlier findings at 25 degrees C [Bahnson et al. (1993) Biochemistry 31, 5503], the F93W mutant shows much less kinetic complexity than the wild-type enzyme between 3 and -35 degrees C. An analysis of noncompetitive deuterium isotope effects and competitive tritium isotope effects leads to the conclusion that the reaction of F93W involves substantial hydrogen tunneling down to -35 degrees C. The effect of methanol on kinetic properties for the F93W mutant was analyzed, showing a dependence of competitive KIEs on the NAD(+) concentration. This indicates a more random bi--bi kinetic mechanism, in comparison to an ordered bi-bi kinetic mechanism in water. Although MeOH also affects the magnitude of the reaction rates and, to some extent, the observed KIEs, the ratio of ln k(H)/k(T) to ln k(D)/k(T) for prim...Continue Reading

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Citations

Oct 2, 2001·Current Opinion in Chemical Biology·V L Schramm
Dec 9, 2008·Journal of the American Chemical Society·Sudhir C Sharma, Judith P Klinman
Jul 22, 2009·Nature Chemical Biology·Zachary D Nagel, Judith P Klinman
Nov 26, 2008·Natural Product Reports·Marc W van der Kamp, Adrian J Mulholland
Mar 24, 2010·Chemical Communications : Chem Comm·Richard LonsdaleAdrian J Mulholland
Jun 27, 2002·European Journal of Biochemistry·Dimitri AntoniouSteven D Schwartz
Jun 15, 2011·Proceedings of the National Academy of Sciences of the United States of America·Zachary D NagelJudith P Klinman
Sep 26, 2013·Proceedings of the National Academy of Sciences of the United States of America·Louis Y P LukRudolf K Allemann
Jul 29, 2006·Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences·Judith P Klinman
Apr 22, 2003·Annual Review of Biochemistry·Daniel A KrautDaniel Herschlag
Feb 7, 2014·Journal of the American Chemical Society·Anna Lewandowska-AndralojcEtsuko Fujita
Jun 12, 2013·Annual Review of Biochemistry·Judith P Klinman, Amnon Kohen
May 18, 2016·The Journal of Chemical Physics·Arieh Warshel, Ram Prasad Bora
Dec 23, 2017·Protein Science : a Publication of the Protein Society·Karthik K ShanmuganathamBryce V Plapp
Aug 10, 2006·Chemical Reviews·Zachary D Nagel, Judith P Klinman

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