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Human ubiquitin-specific cysteine protease 5 (USP5, also known as ISOT and isopeptidase T), an 835-residue multidomain enzyme, recycles ubiquitin by hydrolyzing isopeptide bonds in a variety of unanchored polyubiquitin substrates. Activation of the enzyme’s hydrolytic activity towards ubiquitin-AMC (7-amino-4-methylcoumarin), a fluorogenic substrate, by the addition of free, unanchored mono-ubiquitin suggested an allosteric mechanism of activation by the ZnF-UBP domain (residues 163–291), which binds the substrate’s unanchored diglycine carboxyl tail. By determining the structure of full-length USP5, we discovered the existence of a cryptic ZnF-UBP domain (residues 1–156), which was tightly bound to the catalytic core and was indispensable for catalytic activity. In contrast, the previously characterized ZnF-UBP domain did not contribute directly to the active site; a paucity of interactions suggested flexibility between these two domains consistent with an ability by the enzyme to hydrolyze a variety of different polyubiquitin chain linkages. Deletion of the known ZnF-UBP domain did not significantly affect rate of hydrolysis of ubiquitin-AMC and suggested that it is likely associated mainly with substrate targeting and specificity. Together, our findings show that USP5 uses multiple ZnF-UBP domains for substrate targeting and core catalytic function.

Although the structures of mammalian cytosolic and mitochondrial ALDH have been determined, several differences, mainly functional, between these two 70% identical isozymes remain unexplained. A major difference is the differential effect of Mg(2+) ions that inhibits the cytosolic and activates the mitochondrial isozyme. Here, we have investigated the effect of Mg(2+) ions on each individual kinetic step of ALDH1 and ALDH2. The metal ions were found not to affect either acylation or hydride transfer for either isozyme. The lack of a Mg(2+) ion effect on hydride transfer was further demonstrated with an E399Q mutant of ALDH1 whose rate-limiting step had been changed from NADH dissociation to hydride transfer. The other steps, however, were affected by Mg(2+) ions for both isozymes. The metal ions inhibited NADH dissociation, the rate-limiting step for ALDH1, and enhanced deacylation, the rate-limiting step for ALDH2. Our results indicated that, with both isozymes, Mg(2+) ions tightened the binding of NADH, and by binding to the coenzyme, they increased the nucleophilicity of the nucleophile Cys302. The inhibition of ALDH1 and activation of ALDH2 at pH 7.4 are due to their different rate-limiting steps. Mg(2+) ions affected similarly the NADH activation of the esterase reaction for both isozymes. In contrast, the metal ions affected only the NAD(+) activation of ALDH1. This latter finding and other features described here can be rationalized on the basis of the known three-dimensional structures of the isozymes.

Crystallographic analysis revealed that the nicotinamide ring of NAD can bind with multiconformations to aldehyde dehydrogenase (ALDH) (Ni, L., Zhou, J., Hurley, T. D., and Weiner, H. (1999) Protein Sci. 8, 2784-2790). Electron densities can be defined for two conformations, neither of which appears to be compatible with the catalytic reaction. In one conformation, it would prevent glutamate 268 from functioning as a general base needed to activate the catalytic nucleophile, cysteine 302. In the other conformation, the nicotinamide is too far from the enzyme-substrate adduct for efficient hydride transfer. In this study, NMR and fluorescence spectroscopies were used to demonstrate that NAD and NADH bind to human liver cytosol and mitochondrial ALDH such that the nicotinamide samples a population of conformations while the adenosine region remains relatively immobile. Although the nicotinamide possesses extensive conformational heterogeneity, the catalyzed reaction leads to the stereospecific transfer of hydride to the coenzyme. Mobility allows the nicotinamide to move into position to be reduced by the enzyme-substrate adduct. Although the reduced nicotinamide ring retains mobility after NADH formation, the extent of the motion is less than that of NAD. It appears that after reduction the population of favored nicotinamide conformations shifts toward those that do not interfere with the ability of the enzyme to release the reaction product. In the case of the mitochondrial, but not the cytosolic, enzyme this change in conformational preference is promoted by the presence of Mg2+ ions. Coenzyme conformational mobility appears to be beneficial to catalysis by ALDH throughout the catalytic cycle.