Effect of citrate on the redox properties of cytochrome C and its kinetic and binding behaviour with cytochrome oxidase /

Increasing citrate concentration, at constant ionic strength (30 mM) decreases the rate of cytochrome ~ reduction by ascorbate. This effect is also seen at both high (600 mM) and low (19 mM) ionic strengths, and the Kapp for citrate increases with increasing ionic strength. Citrate binds d both ferri -and ferrocytochrome ~, but with a lower affinity for the latter form (Kox . .red d = 2 mM, Kd = 8 mM) as shown by an equilibrium assay with N,N,N',N', Tetramethyl E- phenylenediamine. The reaction of ferricytochrome ~with cyanide is also altered in the presence of citrate: citrate increases the K~PP for cyanide. Column chromatography of cytochrome ~-cytochrome oxidase mixtures shows citrate increases the dissociation constant of the complex. These results are confirmed in kinetic assays for the "loose"site (Km = 20 pM) only. The effect of increasing citrate observable at the "tight" site (Km = 0.25 pM) is on the turnover number and not on the K . These results suggest a mechanism m where anion binding to cytochrome £ at the tight site affects the equilibrium between two forms of cytochrome c bound cytochrome oxidase: an active and an inactive one.

The investigation of the reduction mechanism of the 5a-reductase enzyme of Penicillium decumbens /

The 5a-reductase of Penicillium decumbens ATCC 10436 was used as a model for the mammalian enzyme to investigate the mechanism of reduction of testosterone to 5adihydrotestosterone . The purpose of this study was to search for specific 5a-reductase inhibitors which antagonize prostate cancer . In a whole-cell biotransformation mode, this organism reduced testosterone (1) to 5a-dihydrosteroids (8) and 5aandrostane- 3, 17-dione (9) in yields of 28% and 37% respectively. Control experiments have shown that 5aandrostane- 3, 17-dione (9) can be produced from the corresponding alcohol (8) in a subsequent reaction separate from that catalysed by the 5a-reductase enzyme . Androst-4- ene-3, 17-dione (2) is reduced to give only (9) with a recovery of 80% The stereochemistry of the reduction was determined by 500 MHz ^H NMR analysis of the products resulting from the deuterium labelled substrates. The results were obtained by an analysis of the NOE difference spectra, double-quantum filtered phase sensitive COSY 2-D spectra, and ^^c-Ir 2-D shift correlation spectra of deuterium labelled products. According to the unambiguous assignment of the signals due to H-4a and H-4Ii in 5a-dihydro steroids, the NMR data show clearly that addition of hydrogen to the 4{5)K bond has occurred in a trans manner at positions 413 and 5a. To Study the reduction mechanism of this enzyme, several substrates were prepared as following; 3-methyleneandrost-4-en- 17fi-ol(3), androst-4-en-17i5-ol(5) , androst-4-en-3ii, 17fi-diol (6) and 4, 5ii-epoxyandrostane-3, 17-dione (7) . Results suggest that this enzyme system requires an oxygen atom at the 3-position of the steroid in order to bind the substrate. Furthermore, the mechanism of this 5a-reductase may proceed via direct addition of hydrogen at the 4,5 position without involvement of a carbonyl group as an intermediate.