A5-Ketosteroids

Die Beobachtung, daB A5-3-Ketosteroide auch bei direkter Einfuh-rung der Probe in das Massenspektrometer nahezu gleiche Spektren geben wie entsprechende A4-3-Ketosteroide, diirfte auf eine Isomeri-sierung noch vor der Ionisation zuriickzufiihren sein 175>. 

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). 

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. 

Figure 2 A model active site for A5-3 ketosteroid isomerase studied using density functional theory. See Ref. 44.

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. 

A6-3-Ketosteroids, 113, 162,299 A14-3-Ketosteroids, 113, 249, 337 A1 s-3-Ketosteroids, 337 A4 -3-Ketosteroids, 113, 337 A5-7-3-Ketosteroids, 337 A 14li-3-K etostero ids. 63, 64, 113 A7-6-Ketosteroids, 361 (3-Ketosulfoxides, 221 Ketoxime acetates, 106 Ketoxime tosylates, 106 Ketoximes, 106, 289 Khellin, 384 Kynurenic acid, 387 Kynurenine yellow, 387... 

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

Hydroxylation. Maume and Horning2 report that trimethylsilyl (TMSi) enol ethers of ketosteroids when irradiated with UV light (dibenzoyl peroxide can also be used) are converted into the trimethylsilyl derivative of an a-hydroxy ketone. Thus the TMSi enol ether of A5- mdrostene-3/3-ol-17-one (1) gives (2) as the chief reaction product. Similarly, A -androstene-3/3,17/3-diol-16-one is converted into... 

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