Structural and mechanistic basis for the activation of a low-molecular weight protein tyrosine phosphatase by adenine.
Although the activation of low-molecular weight protein tyrosine phosphatases by certain purines and purine derivatives was first described three decades ago, the mechanism of this rate enhancement was unknown. As an example, adenine activates the yeast low-molecular weight protein tyrosine phosphatase LTP1 more than 30-fold. To examine the structural and mechanistic basis of this phenomenon, we have determined the crystal structure of yeast LTP1 complexed with adenine. In the crystal structure, an adenine molecule is found bound in the active site cavity, sandwiched between the side chains of two large hydrophobic residues at the active site. Hydrogen bonding to the side chains of other active site residues, as well as some water-mediated hydrogen bonds, also helps to fix the position of the bound adenine molecule. An ordered water was found in proximity to the bound phosphate ion present in the active site, held by hydrogen bonding to N3 of adenine and Odelta1 of Asp-132. On the basis of the crystal structure, we propose that this water molecule is the nucleophile that participates in the dephosphorylation of the phosphoenzyme intermediate. Solvent isotope effect studies show that there is no rate-determining transfer of a solvent-derived proton in the transition state for the dephosphorylation of the phosphoenzyme intermediate. Such an absence of general base catalysis of water attack is consistent with the stability of the leaving group, namely, the thiolate anion of Cys-13. Consequently, adenine activates the enzyme by binding and orienting a water nucleophile in proximity to the phosphoryl group of the phosphoenzyme intermediate, thus increasing the rate of the dephosphorylation step, a step that is normally the rate-limiting step of this enzymatic reaction.
Wang
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S
Departments of Chemistry and Biological Sciences, Purdue University, West Lafayette, IN 47907, USA.
Stauffacher
C V
CV
Van Etten
R L
RL
D000225:Adenine / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003460:Crystallization; D018360:Crystallography, X-Ray; D003903:Deuterium / Q000737:chemistry; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents / Q000737:chemistry; D013329:Structure-Activity Relationship
D020536:Enzyme Activators; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D003903:Deuterium; C094972:LTP1 protein, S cerevisiae; D017027:Protein Tyrosine Phosphatases; D000225:Adenine