Desmolase reaction

Steroid hormones are crucial signal molecules in mammals. (The details of their physiological effects are described in Chapter 34.) Their biosynthesis begins with the desmolase reaction, which converts cholesterol to preg-... 

The other semipurified preparation was a soluble (not sedimentable at 105,000 g for 30 minutes) sonicate described by Simpson and Boyd (1966), which was also used to study the dependency of the desmolase reaction on cytochrome P-450 and the TPNH-cytochrome P-450 reductase composed of a flavoprotein and nonheme iron-protein. The desmolase was found to behave essentially in a similar fashion to the ll)8-hydroxylase system described by Omura et al. (1966). 

The most commonly postulated mechanism for the desmolase reaction has been the suggestion of the enzymatic consecutive reaction sequence cholesterol 20a-hydroxycholesterol — (22R)-20a,22-dihydroxycholesterol pregnenolone (Shimizu et al., 1961, 1962 Constantopoulos and Tchen, 1961). This hypothetical scheme was actually advanced on the basis of three suppositions (1) cholesterol is cleaved between C-20 and C-22, as was discussed in a previous section, (2) 20a-hydroxycholesterol was formed from cholesterol-4- C by a cow adrenocortical homogenate in the presence of 20a-hy-droxycholesterol as a trapping agent (Solomon et al., 1956), and (3) both 20a-hydroxycholesterol and (22R)-20a,22-dihydroxycholesterol are much more efficiently transformed to pregnenolone than is cho-... 

In the isolation of steroids from natural sources one must, of course, consider their stability prior to enzyme inactivation, which usually occurs at the commencement of the isolation procedure, when the tissue is extracted with organic solvents. Kahnt and Neher (1965) could observe in bovine adrenal homogenates only trace amounts of 20a-hydroxycholesterol and (22R)-20,22-dihydroxy-cholesterol, as well as pregnenolone or progesterone (< figlgm). As stated earlier the absence of certain derivatives may be due to either their lack of formation or their rapid disappearance from the system. Until recently, primarily because of the lack of appropriate substrates of high specific activity, quantitative studies on the elucidation of the mechanism of the desmolase reaction could not be performed. The study of Ichii et al. (1967a) appears to entail the assumption of a steady state, for which no proof seems to have been provided. Also,... 

The initial reaction in steroid hormone biosynthesis is catalyzed by desmolase (side-chain cleavage complex), which is found in the mitochondria of steroid-producing tissues (e.g., adrenals, gonads). The reaction is shown in figure 20.22. The product, pregnenolone, is subsequently transferred to the endoplasmic reticulum, where an oxidation of... 

Figure 16.4 Biosynthesis of various classes of steroid hormones. Reaction (A) is catalyzed by a cholesterol desmolase, which oxidizes the cholesterol side chain. Reactions (D) are catalyzed by 21-hydroxylases, which are defective in congenital adrenal hyperplasia. (Reproduced by permission from Schwarz V. A Clinical Companion to Biochemical Studies. Reading Freeman, 1978, p. 94.)...

The conversion of indole to tryptophan has been much more extensively studied. This is brought about by direct reaction of indole and serine under the influence of the enzyme tryptophan desmolase (better named tryptophan synthetase) (302, 853, 854) which requires p5U-idoxal phosphate as a coenzyme (890). The enzyme has been obtained in the cell-free state (890) and partially purified (965) and its genetics in Neurospora studied in detail (966). Zinc appears to play some part in tryptophan desmolase formation or function (628). 

Key to reactions (enzymes) 0, via mevalonate, isopentenyl phosphate, squalene 1, hydroxylase, desmolase (20,22) S, A -3j8-hydroxysteroid dehydrogenase, 5(4)-isomerase S, 21-hydroxylase 4, 17a-hydroxylase 6, ll 3-hydroxylase 6, desmolase (17,20) 7, 17 -hydroxysteroid dehydrogenase 8, 19-hydroxylase 9, aromatization. 

L-Tryptophan synthase, tryptophan desmolase, L-serine hydro-lyase (adding imhleglycerol-phos-phate) (EC 4.2.1.20) the enzyme catalysing the synthesis of L-tryptophan from L-serine and indole 3-glycerol phosphate. T.s. from E. coli (M, 149,000) and other prokaryotes has 02 subunit composition. The enzyme separates easily into monomeric subunit a (also called protein B, M, 29,000) and dimeric subunit 02 (also called protein B Af, of dimer 90,000) when eluted from DEAE cellulose with a sodium chloride gradient. The separated subunits catalyse partial reactions of L-tryptophan synthesis ... 

Tryptophan Degradation. Tryptophan desmolase catalyzes an essentially irreversible reaction. The formation of indole from tryptophan is catalyzed by a very similar but quite distinct enzyme, tryptophanase. ... 

Siibstitution. A modification of the jS-elimination reaction may be substitution of a new group. This has been proposed as the mechanism of action of tryptophan desmolase, in which indole is substituted for the OH of serine. This reaction was found to proceed to a measurable extent in model reactions, in spite of the competing 8-elimination reactions of both serine and tryptophan and other side reactions of indole compounds. Additional substitution reactions of biological significance are the formation of cystathionine from homocysteine and serine and the formation of (Sf-methylcysteine from methyl mercaptan and serine. These reactions are catalyzed by enzymes that require pyridoxal phosphate as a cofactor. 

The enzyme which catalyzes the formation of tryptophan from indole and serine has been named tryptophan desmolase. A cell-free preparation has been obtained from Neurospora mycelium which catalyzes this reaction. Pyridoxal phosphate has been found to be a necessary cofactor. The enzyme has been partially purified by Yanofsky. He found the optimum activity to be at pH 7.8, and confirmed the necessity of pyridoxal phosphate as a coenzyme. Effective inhibitors are Co++, Zn++, CN, hydroxylamine, and tryptophan. 

The final step in the formation of tryptophan is the condensation of indole with serine (Fig. 15, reaction 4). This reaction was demonstrated by Tatum and Bonner ( S4) in Neurospora. The enzyme catalyzing this reaction has been prepared by a number of investigators 2S5-2S7). It was first named tryptophan desmolase 288), and the name indole-serine ligase has also been suggested 286). At present tr3rptophan synthetase is favored. 

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