S-adenosylhomocysteine hydrolase from Acinetobacter calcoaceticus: purification and partial characterization.
S-adenosylhomocysteine hydrolase (SAHase) was purified to homogeneity from the Gram negative strain Acinetobacter calcoaceticus 501. The molecular weight of the native enzyme, estimated by gel permeation, was about 288 KDa, while sodium dodecyl sulfate polyacrylamide gel electrophoresis yielded a relative molecular mass of 48 KDa. The determination of the coenzyme content gave 4 mol of NAD+ and 2 mol of NADH per mol of enzyme. The isoelectric point of native SAHase was at pH 5.1. When assayed in the hydrolytic direction, the Km for S-adenosylhomocysteine and the Vmax of the enzyme for this substrate were 84 microM and 357 mumol/min/mg, respectively; in the synthetic direction, instead, the Km for adenosine and the corresponding Vmax value were 1.6 microM and 37 mumol/min/mg. Substrate analogs were tested for their ability to act as inhibitors and inactivators of the enzyme. Among these compounds, 9-beta-arabinofuranosyl adenine (Ara A) appeared as the most powerful competitive inhibitor (Ki = 18 microM) as well as the strongest time-dependent inactivator. The common feature of all the assayed analogs was the presence of the adenine ring in their molecular structure. It can thus concluded that the presence of the adenine moiety is an essential element in substrate and/or inhibitor interaction with this bacterial enzyme.
Franceschini
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Dipartimento di Scienze e Tecnologie Biomediche e di Biometria, Università dell'Aquila, Italy.
Impagnatiello
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Oratore
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A
Strom
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R
Bozzi
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A
D016954:Acinetobacter calcoaceticus / Q000201:enzymology*; D043383:Adenosylhomocysteinase; D002851:Chromatography, High Pressure Liquid; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000032:analysis; D011684:Purine Nucleosides / Q000494:pharmacology; D012263:Ribonucleosides / Q000494:pharmacology; D013379:Substrate Specificity; D014372:Tubercidin / Q000494:pharmacology; D014740:Vidarabine / Q000494:pharmacology
D011684:Purine Nucleosides; D012263:Ribonucleosides; D009243:NAD; C005248:nebularine; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D014740:Vidarabine; D014372:Tubercidin