Slow binding inhibition of S-adenosylmethionine synthetase by imidophosphate analogues of an intermediate and product.
S-Adenosylmethionine (AdoMet) synthetase catalyzes the only known route of biosynthesis of the primary in vivo alkylating agent. Inhibitors of this enzyme could provide useful modifiers of biological methylation and polyamine biosynthetic processes. The AdoMet synthetase catalyzed reaction converts ATP and L-methionine to AdoMet, PP(i), and P(i), with formation of tripolyphosphate as a tightly bound intermediate. This work describes a nonhydrolyzable analogue of the tripolyphosphate (PPP(i)) reaction intermediate, diimidotriphosphate (O(3)P-NH-PO(2)-NH-PO(3)(5)(-)), as a potent inhibitor. In the presence of AdoMet, PNPNP is a slow-binding inhibitor with an overall inhibition constant (K(i)) of 2 nM and a dissociation rate of 0.6 h(-)(1). In contrast, in the absence of AdoMet PNPNP is a classical competitive inhibitor with a K(i) of 0.5 microM, a slightly higher affinity than PPP(i) itself (K(i) = 3 microM). The imido analogue of the product pyrophosphate, imidodiphosphate (O(3)P-NH-PO(3)(4)(-)) also displays slow onset inhibition only in the presence of AdoMet, with a K(i) of 0.8 microM, compared to K(i) of 250 microM for PP(i). Circular dichroism spectra of the unliganded enzyme and various complexes are indistinguishable indicating that the protein secondary structure is not greatly altered upon complex formation, suggesting local rearrangements at the active site during the slow binding process. A model based on ionization of the bridging -NH- moiety is presented which could account for the potent inhibition by PNP and PNPNP.
Institute for Cancer Research, Fox Chase Cancer Center, 7701 Burholme Avenue, Philadelphia, Pennsylvania 19111, USA.
R S
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Reczkowski
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Markham
D017766:Acid Anhydride Hydrolases / Q000037:antagonists & inhibitors; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D000266:Adenylyl Imidodiphosphate / Q000031:analogs & derivatives / Q000737:chemistry; D019943:Amino Acid Substitution / Q000235:genetics; D001120:Arginine / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics; D011756:Diphosphates / Q000737:chemistry* / Q000494:pharmacology; D004164:Diphosphonates / Q000737:chemistry / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000494:pharmacology; D006868:Hydrolysis / Q000187:drug effects; D007930:Leucine / Q000235:genetics; D008716:Methionine Adenosyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011122:Polyphosphates / Q000737:chemistry* / Q000494:pharmacology
D011756:Diphosphates; D004164:Diphosphonates; D004791:Enzyme Inhibitors; D011122:Polyphosphates; C053489:5'-((homocysteinyl)methyl)adenosine 5'-(beta,gamma-imidotriphosphate); D000266:Adenylyl Imidodiphosphate; C016514:imidodiphosphonic acid; D000255:Adenosine Triphosphate; D001120:Arginine; D008716:Methionine Adenosyltransferase; D017766:Acid Anhydride Hydrolases; C023193:tripolyphosphatase; D007930:Leucine; C005692:triphosphoric acid