3- Ketosteroid isomerase

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. 

Figure J. 10 A5-ketosteroid isomerase-catalyzed conversion of As-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. 

Figure 4-11 The active site of As-ketosteroid isomerase binding to As-androstene-3,17-dione.

Figure 4.15 The local geometry of the A5-androstene-3,17-dione in the complex with As-ketosteroid isomerase.

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. 

Ketosteroid isomerase (lOGX) [30] Asp40 Aspl03 ODl+Tyrl6 OH ... 

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). 

Ketosteroid isomerase (KSI) Produces insoluble fusion protein 125 aa... 

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. 

KuUopulos A, Mildvan AS, Shortle D, Talalay P. Ketosteroid isomerase. Biochemistry 1989 28 149-159. 

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