Steroid isomerase reaction

P450s catalyze a number of other oxidative reactions, on substrates that range from alkanes to complex endogenous molecules such as steroids and fatty acids. Table 10.1 lists many of the oxidative reactions catalyzed by P450s. These enzymes are also known to catalyze non-oxidative dehydrase, reductase and isomerase reactions [15],... 

For several years, she studied chemical reaction mechanisms, especially those involving sulfoxide carbanions and their synthetic applications. Then, she turned to mechanistic enzymology. Her main interests concerned steroid isomerases and cytochrome P-450, vitamin K-dependent carboxylations, and biotin biosynthesis. She contributed to the mechanistic understanding of several enzymes of the pathway, namely, amino-oxopelargo-nate synthase, diaminopelargonate aminotransferase, and more importantly biotin synthase. 

Measurement of Rates of Inhibition. The inhibition of A -3-keto-steroid isomerase activity has been studied in systems containing in a final volume of 500 /il 1 mAf potassium phosphate at pH 7.0/ 5-8 pM crystalline A -3-ketosteroid isomerase of P. testosteroni, and 10-200 pM allenic or acetylenic steroid (steroid added in 20 jal of 1,4-dioxane). The reactions were run at 25° in small polypropylene tubes containing a... 

Application of CBS extrapolations to the A5-ketosteroid isomerase-catalyzed conversion of A5-androstene-3,17-dione to the A4 isomer (Fig. 4.10) provides a test case for extensions to enzyme kinetics. This task requires integration of CBS extrapolations into multilayer ONIOM calculations [56, 57] of the steroid and the active site combined with a polarizable continuum model (PCM) treatment of bulk dielectric effects [58-60], The goal is to reliably predict absolute rates of enzyme-catalyzed reactions within an order of magnitude, in order to verify or disprove a proposed mechanism. 

A great deal of research has been undertaken to determine if the dehydrogena-tion/isomerization reactions are properties of one system or of two separate enzymes (see Ref. 52) most of the evidence suggests that the former is true. Probably three, or even four, substrate-specific isomerases may occur in bovine adrenal cortex which can act on C19, C21 and C27 steroids. Likewise, separate 5-ene-3/3-HSDs may exist in the adrenal cortex for C19 and C21 steroids, because the latter did not compete with C19 steroids for active sites of the enzymes studied. 

The chemistry of the coupling reaction of the allenic secosteroids to isomerase has been under investigation by Penning and Talalay (82, 83, 84) who report that the site of attachment is in the tetrapeptide sequence 55-58. Identification of the actual site has been hampered by a lability of the bound steroid to both low and high pH. 

Tyrosine can be part of low barrier hydrogen bonds in enzymatic reactions. This is snggested for ketosteroid isomerases . A fractionation factor of the COOH proton of Asp-99 (0.34) snpports this ". The phenol proton having a hydrogen bond to the steroid shows a fractionation factor of 0.97. The fractionation factors can be related to the O- O distance . 

Oxo-A -ketosteroid isomerase (KSI), that catalyzes the cofactor-independent tautomeric interconversion of the a,j8- and j8,y-unsaturated 3-oxo-steroids via a dieno-late anion intermediate, has received considerable recent mechanistic and structural scrutiny by Pollack and Mildvan and their coworkers. The reaction catalyzed by KSI is arguably simpler than that catalyzed by TIM the value of the pKa of the unconjugated 5-androstene-3,17-dione is 12.7 whereas that of the conjugated... 

J -3-Keto isomerase catalyzes the isomerization of J -3-ketosteroids to zl -3-ketosteroids by stereospecific transfer of a hydrogen atom from C(4) to C(6). There is considerable evidence that it is the 40- and 6/5-hydrogens that are involved and that the reaction proceeds via an enolic intermediate. A low resolution (6 A) crystal structure determination has been published and the probable steroid-binding site identified via a bound inhibitor, 4-acetoxy-mercuric estradiol. The results of a higher resolution study (2.5 A) combined with the results of NMR studies and analysis of activity of mutant forms of the enzyme have helped to further define the probable active site of the enzyme [64]. 

The distribution of 0 between the hydroxyl and hydroxymethylene functions of 19a and 19b, determined by the fragmentation pattern of these two species during mass spectrometry, could only result from addition of the carboxyl of the enzyme to the a face of the steroid. Had the carboxyl added to the spiro carbon from the jS face, the 0 would have been located in the hydroxymethylene function of 19a. Clearly, if the binding of the oxiranyl steroids is analogous to that of steroid substrates, Asp-38 could not be involved in the normal intramolecular proton transfer associated with the isomerization reaction. However, as recently emphasized by Pollack ei al., the oxiranyl steroids could conceivably bind to the active site in an upsidedown orientation in comparison to that of the substrate steroids (133). In this case, Asp-38 would still be the most probable base involved in the intramolecular proton transfer reaction. Perhaps upsidedown binding accounts for the report that the C-4a hydrogen of 5-androstene-3,17-dione undergoes slow labilization in the presence of the isomerase (134, 135). 

The different conformations of the 4-ene-3-one A-rings of many hormonal steroids have been discussed in Section 3.2 (see Fig. 3.2.3). A 5-ene-3-one isomerase produces these steroids by a stereospecific transfer of a hydrogen atom from C4 to C6. The 4 - and 6P hydrogens are involved, and the reaction proceeds via an enolic intermediate. The isomerase is an elongated dimer in which the steroid is entrapped in a barrel of eight 3-strands (Fig. 9.6.4). 

In the next reaction steps, the enzymes 3)S-hydroxy-A -steroid dehydrogenase, steroid A-isomerase and steroid 17a-hydroxylase are involved. Oxidation at C-3 and double bond migration leads to progesterone, the hormone of the corpus luteum. Afterwards, the C-17 position is hydroxylated to give 17 -hydroxyprogesterone. The reverse reaction sequence is also known and... 

Based on the proposed molecular mechanism of this reaction, a series of acetylenic 5,10-secosteroids has been prepared in the belief that they might serve as substrates for A -3-ketosteroid isomerase. Abstraction of the proton at C-4 by the enzyme should then generate, via an enolic intermediate, the corresponding highly reactive conjugated allenic ketones, which might be expected to react covalently with a nucleophilic amino acid residue at the active site (Scheme 3). This proposal was based on expected conformational similarities between the acetylenic 5,10-seoo-steroids and the normal A -S-ketosteroid substrates for the enzyme. 

Teflon-coated magnetic stirrer. The enzyme activity in a parallel incubation system, containing all components except the steroid, was entirely stable for several hours. Small aliquots (1-20 /il) were removed from the reaction vessels at suitable time intervals (1-3 min) and diluted with 1.0% neutralized bovine serum albumin. The dilutions required to obtain measurable rates in the subsequent assays depended on the degree of inactivation of the enzyme, but were in the range of 2 X 10 - to 2 X 10 -fold. Aliquots of the diluted enzyme were then assayed for isomerase activity at 25° in systems containing in a final volume of 3.0 ml 57.8 fjiM... 

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