Two dehydrogenases : succinic semialdehyde dehydrogenase and retinol dehydrogenase

This work reports studies on two dehydrogenases from mammalian sources. Porcine succinic semialdehyde dehydrogenase was purified to homogeneity from brain while retinol dehydrogenase was partially purified from bovine retina. The purification and characterization of these two enzymes are discussed in detail in this dissertation.

The enzyme succinic semialdehyde dehydrogenase from pig brain was purified 2400-fold by a combination of DEAE-cellulose, hydroxyapatite and AMp-sepharose chromatography. This preparation had a molecular weight of 160,000 and a specific activity of 5.3umole/min mg at 25°C. The enzyme was shown to be composed of four identical subunits of 4l,000 molecular weight, by SDS-polyacrylamide gel electrophoresis and 8M urea isoelectric focusing polyacrylamide gel electrophoresis.

The kinetic properties of this enzyme were examined in detail. The enzyme was found to be irreversible and NAD ^{+ specific. The Km's for succinic semialdehyde and NAD + were 15μM and 310μM respectively. Succinic semialdehyde was also found to be a substrate inhibitor with an inhibition constant of 9OuM. The five carbon semialdehyde, glutaric semialdehyde, was also found to be both a substrate and a substrate inhibitor for succinic semialdehyde dehydrogenase with a Michaelis constant of 3.4uM and an inhibition constant of 3.lmM. The three carbon malonic semialdehyde was neither a substrate nor an inhibitor. With the use of the product inhibitor NADH and dead end inhibitors p-hydroxybenzaldehyde and m-hydroxybenzaldehyde, succinic semialdehyde dehydrogenase was shown to function via a rapid equilibrium random bi bi mechanism.}

Studies of the coupled assay system of 4-aminobutyric acid transaminase and succinic semialdehyde dehydrogenase indicate the two enzymes can function as a valid assay system if the dehydrogenase is in a five fold molar excess of the transaminase. A transient kinetic study of the lag time of the coupled assay as a function of dehydrogenase concentration indicates these two metabolically connected enzymes do not physically interact in solution even with dehydrogenase concentrations as high as O.6uM.

The inhibition of succinic semialdehyde dehydrogenase by carbonyl compounds such as P-pyridoxal and o-phthalaldehyde was investigated in detail. The enzyme is reversibly inhibited by preincubation with Ppyridoxal (300:l mixing molar ratio) at either 25° or 37°C. Reduction with NaBH₄ results in the incorporation of approximately 4 mole of ppyridoxal residues per mole of enzyme. NAD^{+ protects the enzyme against inactivation by P-pyridoxal, whereas the substrate succinic semialdehyde failed to prevent the reaction of P-pyridoxal with lysine residues of the protein. The binding of approximately 10 moles of o-phthalaldehyde per mole of enzyme results in irreversible loss of catalytic activity. The reaction was fast and easily monitored by absorption and fluores-}

cence spectroscopy.

The binding of NADH to succinic semialdehyde dehydrogenase was examined using the fluorescent properties of NADH. The emission spectrum of NADH in the presence of enzyme is found to be enhanced and blue

shifted from that of free NADH indicating the formation of a binary enzyme-NADH complex. .Addition of succinic semialdehyde (10^-4M) to this binary complex brings about further fluorescence enhancement to the binary complex but no additional spectral shift was observed. Addition of succinate (10^{-3M) to the binary enzyme-NADH complex had no effect upon the complexes emission.}

A titration of the emission anisotropy of NADH upon addition to succinic semialdehyde dehydrogenase indicates two moles of NADH bind per mole of enzyme with an affinity of 4&muM.

A titration of enhancement of NADH fluorescence upon addition of NADH to the enzyme also indicates two NADH binding sites with a dissociation constant of 4μM, exist per mole of enzyme. . Initial velocity studies demonstrate NADH is a competitive inhibitor of NAD+ with an inhibition constant of 1.5 x 10^-4M. These observations coupled with the subunit composition and inability to detect any association dissociation phenomena suggests succinic semialdehyde dehydrogenase displays extreme negative cooperativity with respect to NADH.

Retinol dehydrogenase was purified 25 fold from the rod outer segments of bovine retinas by a combination of hydroxyapatite and retinyl sepharose chromatography. The enzyme was found to be revers-

constants for NADp ^{+ and NADPH were 6μM and 4μM respectively, while those of all-trans retinol and all-trans retinal were respectively 9μM and 4μM.}

The enzyme could be partially inactivated with the carbonyl reagent P-pyridoxal. However, contrary to previously reported results, the enzyme could not be inactivated by addition of NaBH, to a mixture of all-trans retinal and partially purified retinol dehydrogenase.

The phospholipid dependence of this enzyme was investigated using phospholipase A2 and phospholipase C. Phospholipase A2 could bring about a 75% inactivation of this enzyme within two minutes of addition. Phospholipase C had little effect on enzymatic activity. Addition of mixed phospholipids to an inactivated preparation of dehydrogenase from which the phospholipase had been removed resulted in a partial restoration of activity.

Retinal dehydrogenase could be inactivated by illumination with visible light when exogenous retinal (10^-5M) had been added to the enzyme preparation, while no inactivation was observed in the absence of retinal. The possibility that this inactivation was due to the production of singlet oxygen by the photosensitizer retinal was substantiated by the observations that the singlet oxygen quencher azide protected against inactivation while D20, which increases the lifetime of singlet oxygen, enhanced inactivation of retinol dehydrogenase.