title: Role of the Substrate Specificity-Defining Residues of Human SIRT5 in Modulating the Structural Stability and Inhibitory Features of the Enzyme.
abstract: Sirtuins are emerging as the key regulators of metabolism and aging, and their potential activators and inhibitors are being explored as therapeutics for improving health and treating associated diseases. Despite the global structural similarity among all seven isoforms of sirtuins (of which most of them catalyze the deacetylation reaction), SIRT5 is the only isoform that catalyzes the cleavage of negatively charged acylated substrates, and the latter feature appears to be encoded by the presence of Tyr102 and Arg105 residues at the active site pocket of the enzyme. To determine the contributions of the above residues in SIRT5 (vis a vis the corresponding residues of SIRT1) on substrate selectivity, inhibition by EX527 and nicotinamide, secondary structural features and thermal stability of the enzymes, we created single and double mutations (viz. Y102A, R105l, and Y102A/R105I) in SIRT5. The kinetic data revealed that while Y102A mutant enzyme catalyzed both deacetylation and desuccinylation reactions with comparable efficiencies, R105I and Y102A/R105I mutant enzymes favored the deacetylase reaction. Like SIRT1, the nicotinamide inhibition of SIRT5 double mutant (Y102A/R105I) exhibited the mixed non-competitive behavior. On the other hand, the desuccinylation reaction of both wild-type and Y102A mutant enzymes conformed to the competitive inhibition model. The inhibitory potency of EX527 progressively increased from Y102A, R105I, to Y102A/R105 mutant enzymes in SIRT5, but it did not reach to the level obtained with SIRT1. The CD spectroscopic data for the wild-type and mutant enzymes revealed changes in the secondary structural features of the enzymes, and such changes were more pronounced on examining their thermal denaturation patterns. A cumulative account of our experimental data reveal mutual cooperation between Y102 and R105 residues in promoting the desuccinylation versus deacetylation reaction in SIRT5, and the overall catalytic feature of the enzyme is manifested via the mutation induced modulation in the protein structure.
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota, United States.
authors.forename: Junru
authors.initials: J
authors.lastname: Yu
authors.affiliation: Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota, United States.
authors.forename: Manas
authors.initials: M
authors.lastname: Haldar
authors.affiliation: Department of Pharmaceutical Sciences, North Dakota State University, Fargo, North Dakota, United States.
authors.forename: Sanku
authors.initials: S
authors.lastname: Mallik
authors.affiliation: Department of Chemistry and Biochemistry, North Dakota State University, Fargo, North Dakota, United States.
authors.forename: D K
authors.initials: DK
authors.lastname: Srivastava
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D055162:Biocatalysis / Q000187:drug effects; D002151:Calorimetry; D002227:Carbazoles / Q000737:chemistry / Q000494:pharmacology; D020134:Catalytic Domain; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009536:Niacinamide / Q000494:pharmacology; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D056564:Sirtuin 1 / Q000235:genetics; D037761:Sirtuins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000187:drug effects; D013696:Temperature
chemical_list: C550547:6-chloro-2,3,4,9-tetrahydro-1H-carbazole-1-carboxamide; D000596:Amino Acids; D002227:Carbazoles; D050505:Mutant Proteins; D009536:Niacinamide; C027681:isonicotinamide; C508544:SIRT5 protein, human; D056564:Sirtuin 1; D037761:Sirtuins