Ketosteroid isomerase and

Benson, A. M., Talalay, P., Keen, J. H., and Jakoby, W. B., Relationship between the soluble glutathione-dependent A -3-ketosteroid isomerase and the glutathione S-transferases of the liver. Proc. Natl. Acad. Sci. U SA. 74, 158-162 (1977). 

There is some experimental evidence that H-bonds with low barriers to proton transfer do occur in enzymatic systems. A H-bond with a low barrier has been detected between a Tyr residue of the enzyme A -S-ketosteroid isomerase and an analog of the intermediate [132], for example. The proton appears to move freely between the Asp and His residues in chymotrypsin... 

Pharmacologically active allenic steroids have already been examined intensively for about 30 years [5], Thus, the only naturally occurring allenic steroid 107 had been synthesized 3 years before its isolation from Callyspongia diffusa and it had been identified as an inhibitor of the sterol biosynthesis of the silkworm Bombyx mori (Scheme 18.34) [86d], At this early stage, allenic 3-oxo-5,10-secosteroids of type 108 were also used for the irreversible inhibition of ketosteroid isomerases in bacteria, assuming that their activity is probably caused by Michael addition of a nucleophilic amino acid side chain of the enzyme at the 5-position of the steroid [103, 104]. Since this activity is also observed in the corresponding /3,y-acetylenic ketones, it can be rationalized that the latter are converted in vivo into the allenic steroids 108 by enzymatic isomerization [104, 105],... 

Several methods have been developed for establishing the MP2 limit for small molecules. We shall compare three of the most important methods, and a recently proposed combination of two of them that achieves a new level of efficiency in obtaining chemically accurate absolute MP2 energy limits. We conclude with a case study of the extension of these approaches to enzyme kinetics, namely the A5-ketosteroid isomerase-catalyzed conversion of A5-androstene-3,17-dione to the A4 isomer. 

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. 

Steroid Systems Labeling of steroid systems, 46, 447 irreversible inhibitors of A -3-ketosteroid isomerase acetylenic and allenic 3-0X0-5,10-secosteroids, 46, 461 labeling of AC3-ketosteroid isomerase by photoexcited steroid ketones, 46, 469. 

This enzyme [EC 5.3.3.1], also called steroid A-isomerase and A -3-ketosteroid isomerase, catalyzes the interconversion of a 3-oxo-A -steroid and a 3-oxo-A -steroid. 

Figure 2.5 Logarithmic scale comparison of k,d and kuncat (= (rnon) for some representative reactions at 25 °C. The length of each vertical bar represents the rate enhancement. (Wolfenden, 2001). ADC arginine decarboxylase ODC orotidine 5 -phosphate decarboxylase STN staphylococcal nuclease GLU sweet potato /3-amylase FUM fumarase MAN mandelate racemase PEP carboxypeptodase B CDA E. coli cytidine deaminase KSI ketosteroid isomerase CMU chorismate mutase CAN carbonic anhydrase.

While true photoaffinity labeling has been achieved with a, J-unsatura-ted ketones (Table 2.1, and above), it should be noted that Benisek and coworkers have observed irreversible inhibition of ketosteroid isomerase from two sources without covalent attachment of the reagent (Ogez et al., 1977 Smith and Benisek, 1980). In each case stoichiometric modification of a residue at the active sites was detected (see Fig. 2.4, Legend). 

Figure 1 Strategy for cloning a peptide-coding sequence (CDS) as tandem repeats in the vector pET31b. The resulting fusion protein, comprising the ketosteroid isomerase (KSI), peptide repeats, and His-tag, is targeted to inclusion bodies. The fusion protein can be recovered and cleaved, in this case, with cyanogen bromide (CNBr) which acts at the methionine (M) residues allowing further separation of pure peptide from the other fusion components. The cleavage by CNBr results in a C-terminal homoserine lactone (hsl) on each peptide monomer.

A 3-Ketosteroid isomerase (3-KSI). This enz)mie catalyses the allylic isomerization of the 5,6 double bond of A5-3-ketosteroids to the 4,5 position by stereospecific intramolecular transfer of a proton. The enz)mie has been isolated from bacteria, and especially the 3-KSIs from Comamoms testosteroni and Pseudomonas putida have been investigated (Smith et al, 1980). The gene coding for the 3-KSI of Pseudomonas putida biot) e B has been cloned and its nucleotide sequence determined (Kim et al, 1994). 

Seidel, S., Kreis, W. and Reinhard, E. (1990) A5-3fJ-Hydroxysteroid dehydrogenase/ A5-A4-ketosteroid isomerase (3fJ-HSD) a possible enzyme of cardiac glycoside biosynthesis, in cell cultures and plants of Digitalis lanata EHRH. Plant Cell Rep., 8, 621-4. 

Smith, S.B., Richards, J.W. and Benisek, W.R (1980) The purification and characterization of A5-3-ketosteroid isomerase from Pseudomonas putida, a cysteine-containing isomerase. /. Biol. Chem., 255, 2678-84. 

Chloro-9-cyclopentyl-8-azapurine inhibited synthesis of DNA, RNA, and protein in E. coli. Blockage of thymine-nucleotide formation was the first effect seen. Alkylation of enzymes by the 6-chloro substituent was suggested as a mechanism. This azapurine inhibited the RNA polymerase from E. coli, but not that from M. lysodeikticus. Formyltetrahydrofolate synthetases, of both mammalian and bacterial origins, were strongly inhibited. The same azapurine, at 0.3 mM, markedly inhibited the steroid-induced synthesis of A -3-ketosteroid isomerase in Pseudomonas testoster-oni ... 

In another variant on the use of paramagnetic species to probe protein structure, Zhao et al.2m determined the secondary structural features of A8-3-ketosteroid isomerase using 3D NMR techniques on the l5N,l3C-labelled protein. This enzyme catalyses the conversion of A5- to A4-3-ketosteroids and is a homodimer of 125 amino acids per subunit. The NMR studies were undertaken on the steroid-bound protein. The amino acids near to the steroid were confirmed by binding a steroid incorporating a spin label and monitoring the disappearance from the 15N- H HSQC spectrum of the cross-peaks associated with these residues. 

Cortisol is synthesized from pregnenolone by two pathways in the fasciculata and reticularis zones of the adrenal gland. For simplicity, only one pathway is shown in Figure 51-6. The enzyme 17a-hydroxylase and the enzyme complex A -3P-hydroxysteroid dehydrogenase A ketosteroid isomerase located in the endoplasmic reticulum will synthesize 17a-hydroxyprogesterone from either 17a-hydroxypregnenolone or progesterone. Hydroxylation of this compound by the... 

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