Substrate specificity of 5′-methylthioadenosine phosphorylase from human prostate

Abstract

5'-Methylthioadenosine phosphorylase was purified approx. 340-fold from human prostate by using affinity chromatography by Hg-coupled Sepharose. The enzyme, responsible for the breakdown of 5'-methylthioadenosine into adenine and methylthioribose 1-phosphate, was partially characterized. The apparent Km for 5'-methylthioadenosine is 25,uM. It is activated by thiols and shows an absolute requirement for phosphate ions. New analogues of 5'-methylthioadenosine were prepared and their activity as substrates or inhibitors of the reaction was investigated. The replacement of the 6-amino group of the adenine moiety by a hydroxy group, as well as the replacement of N-7 by a methinic radical, resulted in an almost complete loss ofactivity. Otherwise the replacement of sulphur by selenium, as well as that of the methyl group by an ethyl one, is compatible with the activity as substrate. The positively charged sulphonium group also prevents catalytic interaction with the enzyme. The inhibitory effect of 5'-methylthiotubercidin (competitive) and 5'-dimethylthioadenosine sulphonium salt (non-competitive) was also demonstrated. The reported results suggest three binding sites between the substrate and the enzyme.

Methylthioinosine Phosphorylase fromPseudomonas aeruginosa. Structure and Annotation of a Novel Enzyme in Quorum Sensing

“…The Asn243Asp mutant and Glu201Gln:Asn243Asp double mutant in human PNP are known to shift the substrate preference in favor of adenosine, a 6-aminopurine substrate and a preferred substrate for MTAP (34). MTAPs prefer 6-aminopurine because of interactions between N1, O6 and N7 of the 6-aminopurine with conserved amino acid residues Ser178 (via water), Asp220 and Asp222 (human MTAP numbering) (29). The ribose binding region of human PNP prefers nucleosides with a 5’-hydroxyl group but not a 5'-methylthio group.…”

Escherichia coli S-adenosylhomocysteine/5′-methylthioadenosine nucleosidase. Purification, substrate specificity and mechanism of action

“…, N6-methyl-3-deaza-adenosylhomocysteine, N6-dimethyl-3-deaza-adenosylhomocysteine, S-inosylhomocysteine, S-cytidylhomocysteine and S-guanosylhomocysteine were kindly given by Dr. R. T. Borchardt, Department of Biochemistry, University of Kansas, Lawrence, KS, U.S.A. 5'-Isobutylthioinosine, 5'-n-butylthioinosine and 5'-methylthioinosine were prepared by enzymic deamination of the corresponding adenosyl derivatives with a non-specific adenosine deaminase from Aspergillus oryzae (Schlenk & ZydeckCwick, 1968;Zappia et al, 1978). 5'-Methylthio-3-deaza-adenosine and 5'-isobutylthio-3-deaza-adenosine were kindly given by Dr. G. Stramentinoli, BioResearch Co., Linate, Italy.…”

Methylthioadenosine

“…In contrast to most enzymes from other thermophiles, the MTA nucleoside phosphorylase was found to be stable at low protein concentrations and at the highest level of purity. At variance with the enzyme from human placenta and prostate gland (240,241), thiol groups appear not to be required for activity.…”

“…AdoHcy was inactive as substrate or inhibitor. Purification of the enzyme from human prostate gave similar results (241). In addition to the earlier experiments, some MTA analogs were tested.…”