On the chemical mechanism of succinic semialdehyde dehydrogenase (GabD1) from Mycobacterium tuberculosis Academic Article uri icon

Overview

MeSH Major

  • Mycobacterium tuberculosis
  • Succinate-Semialdehyde Dehydrogenase

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.

publication date

  • May 2011

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC3094805

Digital Object Identifier (DOI)

  • 10.1016/j.abb.2011.01.023

PubMed ID

  • 21303655

Additional Document Info

start page

  • 90

end page

  • 9

volume

  • 509

number

  • 1