Mechanistic studies of ubiquitin C-terminal hydrolase L1.
Ubiquitin C-terminal hydrolases (UCHs) cleave Ub-X bonds (Ub is ubiquitin and X an alcohol, an amine, or a protein) through a thioester intermediate that is produced by nucleophilic attack of the Cys residue of a Cys-SH/His-Im catalytic diad. We are studying the mechanism of UCH-L1, a UCH that is implicated in Parkinson's disease, and now wish to report our initial findings. (i) Pre-steady-state kinetic studies for UCH-L1-catalyzed hydrolysis of Ub-AMC (AMC, 7-amido-4-methylcoumarin) indicate that k(cat) is rate-limited by acyl-enzyme formation. Thus, K(m) = K(s), the dissociation constant for the Michaelis complex, and k(cat) = k(2), the rate constant for acyl-enzyme formation. (ii) For K(assoc) (=K(s)(-)(1)), DeltaC(p) = -0.8 kcal mol(-)(1) deg(-)(1) and is consistent with coupling between substrate association and a conformational change of the enzyme. For k(2), DeltaS(++) = 0 and suggests that in the E-S, substrate and active site residues are precisely aligned for reaction. (iii) Solvent isotope effects are (D)K(assoc) = 0.5 and (D)k(2) = 0.9, suggesting that the substrate binds to a form of free enzyme in which the active site Cys exists as the thiol. In the resultant Michaelis complex, the diad has tautomerized to ion pair Cys-S(-)/His-ImH(+). Subsequent attack of thiolate produces the acyl-enzyme species. In contrast, isotope effects for association of UCH-L1 with transition-state analogue ubiquitin aldehyde suggest that an alternative mechanistic pathway can sometimes be available to UCH-L1 involving general base-catalyzed attack of Cys-SH by His-Im.
Laboratory for Drug Discovery in Neurodegeneration, Harvard Center for Neurodegeneration and Repair, 65 Landsdowne Street, Fourth Floor, Cambridge, Massachusetts 02139, USA..
April
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Ross L
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Stein
D000215:Acylation; D003374:Coumarins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000378:metabolism; D007700:Kinetics; D008956:Models, Chemical; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013816:Thermodynamics; D025801:Ubiquitin / Q000494:pharmacology; D043222:Ubiquitin Thiolesterase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014452:Ubiquitins / Q000378:metabolism
D003374:Coumarins; D025801:Ubiquitin; D014452:Ubiquitins; C110770:ubiquitin C-terminal 7-amido-4-methylcoumarin; D012965:Sodium Chloride; D006867:Hydrolases; D043222:Ubiquitin Thiolesterase