S-Adenosylhomocysteine Hydrolase (SAHase) catalyzes the reversible hydrolysis of S-adenosylhomocysteine (SAH) to adenosine and homocysteine in an addition-elimination mechanism. This mechanism could follow one of four possible stereochemical pathways. To distinguish between possible pathways, 5'(R)-(5'-('2)H(,1))-adenosine, 5'(S)-(5'-('2)H(,1))-adenosine, 5'(R)-(5'('2)H(,1))-SAH, 5'(S)-(5'('2)H(,1))-SAH, (E)-(5'-('2)H(,1))-4',5'-dehydro-5'-deoxyadenosine, and (Z)-(5'-('2)H(,1))-4',5'-dehydro-5'-deoxyadenosine were chemically synthesized. SAHase was isolated from calf liver. Incubation of the stereospecifically deuterated adenosines and homocysteine with SAHase gave SAH with retention of configuration at C-5'. Addition of homocysteine to the (E) and (Z) isomers of (5'-('2)H(,1))-4',5'-dehydro-5'-deoxyadenosine by SAHase occurred with syn geometry. Therefore, the overall reaction is a syn elimination-syn addition process, indicating a single base mechanism.
A variety of nucleoside analogs have been shown to inactivate SAHase. Several different mechanisms of inactivation have been suggested. However, none of the inactivators appear to be suicide substrates. Analogous to the reaction of 3-decynoyl-S-NAC with (beta)-hydroxy decanoyl dehydrase, an acetylenic derivative of adenosine would be expected to undergo propargylic rearrangement upon activation by SAHase. The synthesis of acetylenic adenosine is described, as well as the radiolabelled 2-('3)H -acetylenic adenosine and 6'-('3)H -acetylenic adenosine. The irreversible inhibition of acetylenic adenosine on SAHase is dependent on both time and inhibitor concentration. The K(,i) was determined to be 173 nM. Binding studies conducted with radiolabelled acetylenic adenosine showed a binding stoichiometry of 4 moles of acetylenic adenosine per mole tetrameric enzyme.
Aristeromycin is one of the better inactivators of SAHase. Binding studies between biosynthetically prepared 2-('3)H -aristeromycin and SAHase were conducted. A binding stoichiometry of 2.5 moles aristeromycin per tetrameric enzyme was determined.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15950 |
| Date | January 1986 |
| Creators | ASKONAS, LESLIE JEAN |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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