Enzymes 3-ketosteroid-5-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. 

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. 

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. 

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. 

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... 

By contrast, kcat/f m for enzymatic catalysis of deprotonation at carbon is not strongly dependent on intrinsic carbon acidity. For example, kcat/ m is close to the diffusion-controlled limit for both the ketosteroid-isomerase-catalyzed deprotonation of the ketone 1 (pK 13) [39] and the triosephosphate-isomerase-catalyzed deprotonation of the ketone 3 (pK 18) [40]. An extreme example is the small difference in the values of kcat/Km = 3 x 10 and 1.4 x 10 s for enzyme-... 

Fig. 3 A modified Wolfenden plot showing kcat/KM> kcat> and kuncat for a selection of enzymes and antibodies Staphylococcal nuclease (STN), adenosine deaminase (ADA), cytidine deaminase (CDA), alanylalanine transpeptidase (PTE), carboxypeptidase A (CPA), ketosteroid isomerase KSI)y triose phosphate isomerase (TIM), chorismate mutase (CMC), carbonic anhydrase (CAN), cyclophilin (CYC)...

Although cross-axis CPCs yield less efficient separations than the type-J multilayer CPC, they provide more stable retention of the stationary phase and are therefore useful for large-scale preparative separations with polar solvent systems. These are especially useful for the purification of proteins with aqueous-aqueous polymer phase systems composed of PEG and potassium phosphate. The crossaxis CPC has been used for the purification of various enzymes including choline esterase, ketosteroid isomerase, purine nucleoside phosphorylase, lactic acid dehydrogenase, uridine phosphorylase (see Proteins Cross-Axis Coil Planet Centrifuge Separation, p. 1935). 

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. 

Fig. 6.8 Residues in the active site of A -3-ketosteroid isomerase from Escherichia colt [111]. Aspartic acid (D) residues 38 and 99, tyrosines (7) 14, 30 and 55, and atoms 3-6 of a bound steroid substrate are labeled. (Add 2 to the residue numbers to get those in the widely studied enzyme from Pseudomonas piaida.) Dotted lines indicate likely hydrogen htnids. The enzyme catalyzes rearrangement of the C5-C6 double bond to C4-C5 by facilitating enolization of the keto group and transfer of a proton from C4 to C6, probably via D38. The shifted C=0 stretching frequencies of bound 19-norleslosleione point to a stnaig local electric field that favors the enolization [106]...

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... 

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