Purification and kinetic characterization of pickerel liver alcohol dehydrogenase with dual coenzyme specificity

1993 ◽  
Vol 71 (9-10) ◽  
pp. 421-426 ◽  
Author(s):  
Loola S. Al-Kassim ◽  
C. Stan Tsai
Keyword(s):  
Alcohol Dehydrogenase ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Resonance Spectroscopy ◽  
Coenzyme Specificity ◽  
Liver Alcohol Dehydrogenase ◽  
Steady State Kinetic ◽  
Dehydrogenase Isozyme ◽  
State Kinetic

A major alcohol dehydrogenase isozyme that displays dual coenzyme specificity has been purified from pickerel liver by ion-exchange, gel filtration, and affinity chromatographic procedures. The purified enzyme is chromatographically and electrophoretically homogeneous. It is dimeric and possesses common physical properties shared by other liver alcohol dehydrogenases. Phosphorus-31 nuclear magnetic resonance spectroscopy demonstrates that NADP+ binds to two coenzyme sites of the pickerel enzyme. Steady-state kinetic studies suggest that pickerel liver alcohol dehydrogenase catalyzes NAD(P)+-linked ethanol oxidation via a random pathway. While the NADP+ reduction involves the formation of an abortive complex at high NADP+ concentrations, the NAD+ reduction at low NAD+ concentrations follows an ordered Bi-Bi mechanism with NAD+ being the leading reactant.Key words: purification, pickerel liver, alcohol dehydrogenase.

scholarly journals Overexpression, Purification, and Characterization of the Cloned Metallo-β-Lactamase L1 fromStenotrophomonas maltophilia

1998 ◽  
Vol 42 (4) ◽  
pp. 921-926 ◽  
Author(s):  
Michael W. Crowder ◽  
Timothy R. Walsh ◽  
Linda Banovic ◽  
Margaret Pettit ◽  
James Spencer
Keyword(s):  
Molecular Mass ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Laser Desorption Ionization ◽  
Purification And Characterization ◽  
Flight Mass Spectrometry ◽  
Steady State Kinetic ◽  
Cephalosporin Antibiotics ◽  
State Kinetic

ABSTRACT The metallo-β-lactamase L1 from Stenotrophomonas maltophilia was cloned, overexpressed, and characterized by spectrometric and biochemical techniques. Results of metal analyses were consistent with the cloned enzyme having 2 mol of tightly bound Zn(II) per monomer. Gel filtration chromatography demonstrated that the cloned enzyme exists as a tightly held tetramer with a molecular mass of ca. 115 kDa, and matrix-assisted laser desorption ionization and time-of-flight mass spectrometry indicated a monomeric molecular mass of 28.8 kDa. Steady-state kinetic studies with a number of diverse penicillin and cephalosporin antibiotics demonstrated that L1 effectively hydrolyzes all tested compounds, withk cat/Km values ranging between 0.002 and 5.5 μM−1 s−1. These characteristics of the recombinant enzyme are contrasted to those previously reported for metallo-β-lactamases isolated directly fromS. maltophilia.

Multifunctional activities of pickerel liver alcohol dehydrogenase

1993 ◽  
Vol 71 (9-10) ◽  
pp. 427-431 ◽  
Author(s):  
C. Stan Tsai ◽  
Loola S. Al-Kassim
Keyword(s):  
Alcohol Dehydrogenase ◽  
Kinetic Studies ◽  
Regulatory Function ◽  
Amino Acid Residues ◽  
Liver Alcohol Dehydrogenase ◽  
Dead End ◽  
Steady State Kinetic ◽  
Esterolytic Activity ◽  
Hydrolysis Of Esters ◽  
Hydrolysis Of

A major isozyme of pickerel liver alcohol dehydrogenase has been purified to homogeneity. The enzyme, in addition to catalyze NAD(P)+-linked dehydrogenation of alcohols, also mediates dismutation of aldehydes and hydrolysis of esters. Steady-state kinetic studies and chemical modifications of the pickerel liver enzyme with respect to its esterolytic and dismutative activities were carried out. Pickerel liver alcohol dehydrogenase catalyzes hydrolyses of p-nitrophenyl esters via a Uni-Bi mechanism, with alkanoic acids as the last product released. Modifications of Cys and Lys suppress the esterolytic activity. A random mechansim with the formation of dead-end complexes is implicated for the dismutation of octanal catalyzed by pickerel liver alcohol dehydrogenase. Two amino acid residues, Cys and His, are involved in the dehydrogenation as well as dismutation reactions. The present study identifies a regulatory function of Lys for the multifunctional activities of liver alcohol dehydrogenase. When the Lys residue is specifically glucosylated, the dehydrogenase activity increases. Its esterase activity decreases, while the dismutase activity remains unchanged.Key words: multifunctionality, pickerel liver, alcohol dehydrogenase.

Kinetic Studies with Liver Alcohol Dehydrogenase*

Biochemistry ◽  
1965 ◽  
Vol 4 (11) ◽  
pp. 2442-2451 ◽  
Author(s):  
Craig C. Wratten ◽  
W. W. Cleland
Keyword(s):  
Alcohol Dehydrogenase ◽  
Kinetic Studies ◽  
Liver Alcohol Dehydrogenase
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