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PubMed1977

Adenosylhomocysteinase from yellow lupin seeds. Purification and properties.

Adenosylhomocysteinase from yellow lupin seeds (Lupinus luteus) has been purified to homogeneity. Active enzyme, Mr = 110000, consists of two probably identical subunits with Mr = 55000 as judged by gel filtration and dodecyl sulphage/polyacrylamide gel electrophoresis in the presence of 2-mercaptoethanol. The isoelectric point of the enzyme was shown to be 4.9 +/- 0.1. It was demonstrated by disc and pore gradient electrophoresis that the most purified fraction formed multimers. The enzyme shows optimum activity at pH 8.5-9.0. Km values are 2.3 micrometer, 4.6 mM and 12 micrometer for adenosine, DL-homocysteine and S-adenosyl-L-homocysteine, respectively. The energy of activation for S-adenosylhomocysteine synthesis was estimated as 14.4 kcal/mol (60.2 kJ/mol) and temperature coefficient as 2.4. The equilibrium constant for the hydrolysis of S-adenosylhomocysteine amounts to 5 X 10(-7) M. Anti-sulfhydryl reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide acted as irreversible inhibitors. The enzyme exhibits high specificity for homocysteine whereas some of the rare nucleosides tested could substitute for adenosine.

Hydrolases↗
PubMed1979

The mechanism of action of S-adenosylhomocysteinase.

S-Adenosylhomocysteinase catalyzes the reversible hydrolysis of S-adenosyl-L-homocysteine (AdoHcy) to adenosine and L-homocysteine without added cofactors. A mechanism is proposed which involves oxidation of the 3'-hydroxyl group of AdoHcy by enzyme-bound NAD+. Following oxidation, L-homocysteine is eliminated, alpha-beta, to give 3'-keto-4'-5'-dehydroadenosine. This compound reacts with water in a Michael type addition to form 3'-ketoadenosine which is then reduced to adenosine. This mechanism is supported by these facts. 1) The enzyme contains 1 tightly bound NAD+ per subunit. Upon addition of substrate, this NAD is converted to NADH. 2) The enzyme catalyzes the exchange of the 4'-proton of substrate with solvent. This exchange is an integral part of the catalytic mechanism. 3) The hydrolysis of [4'-2H]S-adenosyl-L-homocysteine has a Vmax isotope effect of 1.44. This provides additional evidence that cleavage of the C-4' C-H bond is a step on the reaction pathway. 4) 4',5'-Dehydroadenosine is oxidized by the enzyme, then converted into adenosine or into AdoHcy in the presence of L-homocysteine. 5) An adenosine analog, 5'-deoxyadenosine, is oxidized by the enzyme to yield 3'-keto-5'-deoxyadenosine, and an analog of the proposed intermediate, 3'-ketoadenosine. 6) The enzyme catalyzes the exchange of the C-4' proton of 5'-deoxyadenosine. Since the enzyme catalyzes proton abstraction without OH elimination, it was concluded that the elimination of H2O from adenosine proceeds by a carbanion mechanism and not by a concerted elimination. Substrate analogs in which the 5'-OH group of adenosine is replaced by -F, -Cl, or -SMe are not substrates for the enzyme.

Animals↗
PubMed1976

Mechanism for enzymatic thioether formation. Mechanism of action of S-adenosylhomocysteinase.

Homogeneous S-adenosylhomocysteinase contains tightly bound NAD+. This NAD+ is not dissociable under nondenaturing conditions but can be removed by heat or acid denaturation. Addition of adenosine to the enzyme causes an increase in the absorption at 327 nm which we attribute to NADH formation. The enzyme also catalyzes the rapid exchange of the adenosine 4'-proton with solvent water. A mechanism is proposed for the reversible hydrolysis of S-adenosyl-L-homocysteine involving oxidation of position 3' of adenosine followed by alpha-beta elimination of L-homocysteine to give 3'-deto-4',5'-dehydro-5'-deoxyadenosine. This compound reacts with water in a Michael-type addition to form 3'-keto adenosine which is then re-reduced to adenosine.

Animals↗
PubMed1977

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.

Animals↗
PubMed1979

Sequestration of adenosine in crude extract from mouse liver and other tissues.

Adenosine (1 microM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Saebø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333--340). These data point to a role of this protein in the sequestration of adenosine in crude extract. The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed. The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 microM. The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract. The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.

Adenosine↗
PubMedDate not supplied

[The mechanism of action of S-methylmethionine].

S-methyl methionine was found to increase activity of S-adenosyl-L-homocysteine hydrolase, selectively methylating the released homocysteine. This decreased the inhibitory effect of S-adenosyl-L-homocysteine and stimulated methylation processes.

Adenosylhomocysteinase↗