Kinetic mechanisms of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.

The kinetic properties of two cholesterol oxidases, one from Brevibacterium sterolicum (BCO) the other from Streptomyces hygroscopicus (SCO) were investigated. BCO works via a ping-pong mechanism, whereas the catalytic pathway of SCO is sequential. The turnover numbers at infinite cholesterol and oxygen concentrations are 202 s-1 and 105 s-1 for SCO and BCO, respectively. The rates of flavin reduction extrapolated to saturating substrate concentration, under anaerobic conditions, are 235 s-1 for BCO and 232 s-1 for SCO (in the presence of 1% Thesit and 10% 2-propanol). With reduced SCO the rate of Delta5-6-->Delta4-5 isomerization of the intermediate 5-cholesten-3-one to final product is slow (0.3 s-1). With oxidized SCO and BCO the rate of isomerization is much faster ( approximately 300 s-1), thus it is not rate-limiting for catalysis. The kinetic behaviour of both reduced COs towards oxygen is unusual in that they exhibit apparent saturation with increasing oxygen concentrations (extrapolated rates approximately 250 s-1 and 1.3 s-1, for BCO and SCO, respectively): too slow to account for catalysis. For BCO the kinetic data are compatible with a step preceding the reaction with oxygen, involving interconversion of reactive and nonreactive forms of the enzyme. We suggest that the presence of micelles in the reaction medium, due to the necessary presence of detergents to solubilize the substrate, influence the availability or reactivity of oxygen towards the enzyme. The rate of re-oxidation of SCO in the presence of product is also too slow to account for catalysis, probably due to the impossibility of producing quantitatively the reduced enzyme-product complexes.

Characterization of cholesterol oxidase from Streptomyces hygroscopicus and Brevibacterium sterolicum.

The FAD-containing enzyme cholesterol oxidase catalyzes the oxidation and isomerization of 3beta-hydroxysteroids having a trans double bond at delta5-delta6 of the steroid ring backbone to the corresponding delta4-3-ketosteroid. Two representative enzymes of this family, namely cholesterol oxidase from Streptomyces hygroscopicus (SCO) and the recombinant enzyme from Brevibacterium sterolicum (BCO) expressed in Escherichia coli, have been characterized herein in their chemical, physical, and biochemical properties. In the native form, both enzymes are monomeric (55 kDa), acidic (pI 4.4-5.1) and contain oxidized FAD (peaks in the 370-390-nm and 440-470-nm regions). Marked differences exist between the oxidized, reduced, and (red) anion semiquinone spectra of the two enzymes, suggesting substantial differences in the flavin microenvironment. Both enzymes form reversibly flavin N(5)-sulfite adducts via measurable k(on) and k(off) steps. BCO has a higher affinity for sulfite (Kd approximately 0.14 mM) compared to SCO (approximately 24 mM). This correlates well with the midpoint redox potentials of the bound flavin, which in the case of BCO are about 100 mV more positive than for SCO. Both enzymes show a high pKa (approximately 11.0) for the N(3) position of FAD. With both enzymes, the rearrangement of 5-cholesten-3-one to 4-cholesten-3-one is not rate limiting indicating that the rate-limiting step of the overall reaction is not the isomerization. The absence of the double bond in the steroid molecule does not significantly affect turnover and affinity for the substrate, whereas both these parameters are affected by a decreasing length of the substrate C17 chain.

6-Pyruvoyl tetrahydropterin synthase assay in extracts of cultured human cells using high-performance liquid chromatography with fluorescence detection of biopterin.

An assay for 6-pyruvoyl tetrahydropterin synthase, the second enzyme in the conversion of guanosine triphosphate into tetrahydrobiopterin, has been developed. Cell extracts were incubated with enzymatically prepared dihydroneopterin triphosphate (80 microM) in the presence of Mg2+ (12 mM), excess sepiapterin reductase (EC 1.1.1.153) (2 nmol/min) and NADPH (2 mM). 6-Pyruvoyl tetrahydropterin, the product of the reaction, was thus converted into tetrahydrobiopterin. After oxidation of the reduced biopterin derivatives in acidic iodine solution, biopterin was enriched and separated from the abundant neopterin phosphates by solid-phase extraction on a strong cation exchanger. Biopterin was then directly eluted on a reversed-phase liquid chromatographic column and detected fluorimetrically using excitation at 353 nm and emission at 438 nm. The biopterin concentrations formed by the coupled enzyme reaction increased linearly with incubation times up to 90 min. The assay allows the quantification of 6-pyruvoyl tetrahydropterin synthase in cultured human cells.