Mechanism of Aldehyde Oxidation Catalyzed by Horse Liver Alcohol Dehydrogenase

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• 作者：Leif P. Olson ; Jia Luo ; Ö ; rn Almarsson ; and Thomas C. Bruice
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：July 30, 1996
• 年：1996
• 卷：35
• 期：30
• 页码：9782 - 9791
• 全文大小：608K
• 年卷期：v.35,no.30(July 30, 1996)
• ISSN：1520-4995

文摘

The mechanism of oxidation of benzaldehyde to benzoicacid catalyzed by horse liver alcoholdehydrogenase (HLADH) has been investigated using the HLADH structureat 2.1 Å resolution withNAD⁺ and pentafluorobenzyl alcohol in the active site[Ramaswamy et al. (1994) Biochemistry 33,5230-5237]. Constructs for molecular dynamics (MD) investigations withHLADH were obtained by a best-fitsuperimposition of benzaldehyde or its hydrate on the pentafluorobenzylalcohol bound to the active siteZn(II) ion. Equilibrium bond lengths, angles, and dihedralparameters for Zn(II) bonding residues His67,Cys46, and Cys174 were obtained from small-molecule X-ray crystalstructures and an ab initio-derivedparameterization of zinc in HLADH [Ryde, U. (1995) Proteins:Struct., Funct., Genet. 21, 40-56].Dynamic simulations in CHARMM were carried out on the followingthree constructs to 100 ps: (MD1)enzyme with NAD⁺, benzaldehyde, and zinc-ligatedHO^- in the active site; (MD2) enzyme withNAD⁺and hydrated benzaldehyde monoanion bound to zinc via thepro-R oxygen, with a proton residing onthepro-S oxygen; and (MD3) enzyme withNAD⁺ and hydrated benzaldehyde monoanion bound to zincviathe pro-S oxygen, with a proton residing on thepro-R oxygen. Analyses were done of 800sampleconformations taken in the last 40 ps of dynamics. Structures fromMD1 and MD3 were used to definethe initial spatial arrangements of reactive functionalities forsemiempirical PM3 calculations. UsingPM3, model systems were calculated of ground states and some transitionstates for aldehyde hydration,hydride transfer, and subsequent proton shuttling. Withbenzaldehyde and zinc-bound hydroxide ion inthe active site, the oxygen of Zn(II)-OH resided at a distanceof 2.8-5.5 Å from the aldehyde carbonylcarbon during the dynamics simulation. This may be compared to thePM3 transition state for attack ofthe Zn(II)-OH oxygen on the benzaldehyde carbonyl carbon, whichhas an O···C distance of 1.877 Å.HLADH catalysis of the aldehyde hydration would require verylittle motion aside from that in the groundstate. Two simulations of benzaldehyde hydrate ligated to zinc(MD2 and MD3) both showed closeapproach of the aldehyde hydrate hydrogen to NAD⁺ C4,varying from 2.3 to 3.3 Å, seemingly favorablefor the hydride transfer reaction. The MD2 configuration does notallow proton shuttling. On the otherhand, when the pro-S oxygen is ligated to zinc(MD3), the proton on the pro-R oxygen averages2.09 Åfrom the hydroxyl oxygen of Ser48 such that initiation of shuttling ofprotons via Ser48 to the ribose2'-hydroxyl oxygen to the 3'-hydroxyl oxygen to His51 nitrogen issterically favorable. PM3 calculationssuggest that this proton shuttle represents a stepwise reaction whichoccurs subsequent to hydride transfer.The PM3 transition state for hydride transfer based on the MD3configuration has the transferring hydride1.476 Å from C4 of NAD⁺ and 1.433 Å from the aldehyde-carbon.