title: On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis.
abstract: Succinic semialdehyde dehydrogenases (SSADHs) are ubiquitous enzymes that catalyze the NAD(P)+-coupled oxidation of succinic semialdehyde (SSA) to succinate, the last step of the γ-aminobutyrate shunt. Mycobacterium tuberculosis encodes two paralogous SSADHs (gabD1 and gabD2). Here, we describe the first mechanistic characterization of GabD1, using steady-state kinetics, pH-rate profiles, ¹H NMR, and kinetic isotope effects. Our results confirmed SSA and NADP+ as substrates and demonstrated that a divalent metal, such as Mg²+, linearizes the time course. pH-rate studies failed to identify any ionizable groups with pK(a) between 5.5 and 10 involved in substrate binding or rate-limiting chemistry. Primary deuterium, solvent and multiple kinetic isotope effects revealed that nucleophilic addition to SSA is very fast, followed by a modestly rate-limiting hydride transfer and fast thioester hydrolysis. Proton inventory studies revealed that a single proton is associated with the solvent-sensitive rate-limiting step. Together, these results suggest that product dissociation and/or conformational changes linked to it are rate-limiting. Using structural information for the human homolog enzyme and ¹H NMR, we further established that nucleophilic attack takes place at the Si face of SSA, generating a thiohemiacetal with S stereochemistry. Deuteride transfer to the Pro-R position in NADP+ generates the thioester intermediate and [4A-²H, 4B-¹H] NADPH. A chemical mechanism based on these data and the structural information available is proposed.
authors.affiliation: Department of Microbiology and Immunology, Weill Medical College of Cornell University, 1300 York Ave. New York, NY 10065, USA. lsd2001@med.cornell.edu
authors.forename: Luiz Pedro S
authors.initials: LP
authors.lastname: de Carvalho
authors.forename: Yan
authors.initials: Y
authors.lastname: Ling
authors.forename: Chun
authors.initials: C
authors.lastname: Shen
authors.forename: J David
authors.initials: JD
authors.lastname: Warren
authors.forename: Kyu Y
authors.initials: KY
authors.lastname: Rhee
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000737:chemistry / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D016415:Sequence Alignment; D013237:Stereoisomerism; D013379:Substrate Specificity; D050644:Succinate-Semialdehyde Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D009249:NADP; D005680:gamma-Aminobutyric Acid; D050644:Succinate-Semialdehyde Dehydrogenase; C009338:succinic semialdehyde