Purification and partial characterization of pyridoxal dehydrogenase from porcine liver

Author

Yongzhang Luo

Date of Award

8-1989

Degree Type

Thesis

Degree Name

Master of Science

Major Professor

Jorge E. Churchich

Committee Members

John W. Koontz, Elizabeth E. Howell

Abstract

Aldehyde dehydrogenase ( ALDH ), an NAD-dependent enzyme which oxidizes acetaldehyde to acetate is localized in cytosol, mitochondria and microsomes of mammalian liver cells. The oxidation of aldehyde substrates to carbonic acids is catalyzed by different enzymes. The reaction is highly exergonic and, therefore, irreversible even in dehydrogenase reactions with NAD as electron/hydrogen acceptor. Knowledge of the catabolism of pyridoxal, which is one kind of aromatic aldehyde and one of the derivatives of Vitamin B₆ in mammalian tissues is still incomplete. An aldehyde dehydrogenase which oxidizes pyridoxal to pyridoxic acid has been postulated to be the key enzyme inactivation rate of Vitamin B₆. Two assay methods, fluorometric and spectrophotometric methods, were developed in the identification of pyridoxal dehydrogenase from porcine liver. It has been detected that this enzyme catalyzes the conversion of PL and NAD to PA and NADH, respectively and the reverse reaction does not occur. PD was purified to 1300 fold from porcine liver and its molecular weight was estimated by gel to be 123,000 daltons. The pH optimum lies between 8.5-9.0. It has been found that PD shows very high specificity for PL and does net oxidize benzaldehyde and salicylaldehyde which are believed to be the common substrates of aldehyde dehydrogenases. Thus, PD is not a general aldehyde dehydrogenase. The K_m values for PL and NAD were determined to be 2.46 uM and 75.7 uM at PH 7.5, respectively. Inhibition studies show that both NADH and 1,10-phenanthroline are competitive with respect to NAD. 4,7-phenanthroline also shows almost the same inhibition to PD with 1,10-phenanthroline, indicating that there is no zinc ion in the active site of PD. The uncompetitive inhibition pattern of NADH with respect to PL indicates the reaction catalyzed by PD follows an ordered sequential mechanism. The stereochemistry of hydride transfer by PD was shown to be pro-S.

Recommended Citation

Luo, Yongzhang, "Purification and partial characterization of pyridoxal dehydrogenase from porcine liver. " Master's Thesis, University of Tennessee, 1989.
https://trace.tennessee.edu/utk_gradthes/13006