scholarly journals Substrate specificity of sheep liver sorbitol dehydrogenase

1998 ◽  
Vol 333 (3) ◽  
pp. 847-848
Author(s):  
R. I. LINSTAD ◽  
P. KÖLL ◽  
J. S. McKINLEY-McKEE
Keyword(s):  
Substrate Specificity ◽  
Sorbitol Dehydrogenase ◽  
Sheep Liver

scholarly journals Substrate specificity of sheep liver sorbitol dehydrogenase

1998 ◽  
Vol 330 (1) ◽  
pp. 479-487 ◽  
Author(s):  
I. Rune LINDSTAD ◽  
Peter KÖLL ◽  
John S. McKINLEY-McKEE
Keyword(s):  
Substrate Specificity ◽  
Ternary Complex ◽  
Kinetic Mechanism ◽  
Sorbitol Dehydrogenase ◽  
Hydroxyl Group ◽  
Enzyme Active Site ◽  
Sheep Liver ◽  
Rate Limiting Step ◽  
Steady State Kinetics ◽  
Rate Limiting

The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

scholarly journals Catalytic mechanism of Zn2+-dependent polyol dehydrogenases: kinetic comparison of sheep liver sorbitol dehydrogenase with wild-type and Glu154→Cys forms of yeast xylitol dehydrogenase

2007 ◽  
Vol 404 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Mario Klimacek ◽  
Heidemarie Hellmer ◽  
Bernd Nidetzky
Keyword(s):  
Rate Constant ◽  
Catalytic Mechanism ◽  
Isotope Effects ◽  
Bound Water ◽  
Cysteine Residue ◽  
Xylitol Dehydrogenase ◽  
Sorbitol Dehydrogenase ◽  
Wild Type ◽  
Ph Profile ◽  
Sheep Liver

Co-ordination of catalytic Zn2+ in sorbitol/xylitol dehydrogenases of the medium-chain dehydrogenase/reductase superfamily involves direct or water-mediated interactions from a glutamic acid residue, which substitutes a homologous cysteine ligand in alcohol dehydrogenases of the yeast and liver type. Glu154 of xylitol dehydrogenase from the yeast Galactocandida mastotermitis (termed GmXDH) was mutated to a cysteine residue (E154C) to revert this replacement. In spite of their variable Zn2+ content (0.10–0.40 atom/subunit), purified preparations of E154C exhibited a constant catalytic Zn2+ centre activity (kcat) of 1.19±0.03 s−1 and did not require exogenous Zn2+ for activity or stability. E154C retained 0.019±0.003% and 0.74±0.03% of wild-type catalytic efficiency (kcat/Ksorbitol=7800±700 M−1· s−1) and kcat (=161±4 s−1) for NAD+-dependent oxidation of sorbitol at 25 °C respectively. The pH profile of kcat/Ksorbitol for E154C decreased below an apparent pK of 9.1±0.3, reflecting a shift in pK by about +1.7–1.9 pH units compared with the corresponding pH profiles for GmXDH and sheep liver sorbitol dehydrogenase (termed slSDH). The difference in pK for profiles determined in 1H2O and 2H2O solvent was similar and unusually small for all three enzymes (≈+0.2 log units), suggesting that the observed pK in the binary enzyme–NAD+ complexes could be due to Zn2+-bound water. Under conditions eliminating their different pH-dependences, wild-type and mutant GmXDH displayed similar primary and solvent deuterium kinetic isotope effects of 1.7±0.2 (E154C, 1.7±0.1) and 1.9±0.3 (E154C, 2.4±0.2) on kcat/Ksorbitol respectively. Transient kinetic studies of NAD+ reduction and proton release during sorbitol oxidation by slSDH at pH 8.2 show that two protons are lost with a rate constant of 687±12 s−1 in the pre-steady state, which features a turnover of 0.9±0.1 enzyme equivalents as NADH was produced with a rate constant of 409±3 s−1. The results support an auxiliary participation of Glu154 in catalysis, and possible mechanisms of proton transfer in sorbitol/xylitol dehydrogenases are discussed.

Zinc environment in sheep liver sorbitol dehydrogenase

Biochemistry ◽  
1989 ◽  
Vol 28 (18) ◽  
pp. 7257-7262 ◽  
Author(s):  
Martinus C. Feiters ◽  
Jonathan Jeffery
Keyword(s):  
Sorbitol Dehydrogenase ◽  
Sheep Liver
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