Selective determination of the catalytic cysteine pKa of two-cysteine succinic semialdehyde dehydrogenase from Acinetobacter baumannii using burst kinetics and enzyme adduct formation

Abstract

Succinic semialdehyde dehydrogenase (SSADH) from Acinetobacter baumannii (Ab) catalyzes the oxidation of succinic semialdehyde (SSA). This enzyme has two conserved cysteines (Cys289 and Cys291) and preferentially uses NADP⁺ over NAD⁺ as a hydride acceptor. Steady-state kinetic analysis showed that AbSSADH has the highest catalytic turnover (137 s^-1 ) and can tolerate SSA inhibition the most (< 500 μm) among all SSADHs reported. Alanine substitutions of the two conserved cysteines indicated that Cys291Ala has ~ 65% activity compared with the wild-type enzyme while Cys289Ala is inactive, suggesting that Cys289 is the active residue participating in catalysis. Pre-steady-state kinetics showed for the first time burst kinetics for NADPH formation in SSADH, indicating that the rate-limiting step is associated with steps that occur after the hydride transfer. As the magnitude of burst kinetics represents the amount of NADPH formed during the first turnover, it is directly dependent on the amount of the deprotonated form of cysteine. The pK_a of Cys289 was calculated from a plot of the burst magnitude vs pH as 7.4 ± 0.2. The Cys289 pK_a was also measured based on the ability of AbSSADH to form an NADP-cysteine adduct, which can be detected by the increase of absorbance at ~ 330 nm as 7.9 ± 0.2. The lowering of the catalytic cysteine pK_a by 0.6-1 unit renders the catalytic thiol more nucleophilic, which facilitates AbSSADH catalysis under physiological conditions. The methods established herein can specifically measure the active site cysteine pK_a without interference from other cysteines. These techniques may be useful for studying ionization state of other cysteine-containing aldehyde dehydrogenases.

Enzyme: Succinic semialdehyde dehydrogenase, EC1.2.1.24.

Keywords: NADP-cysteine adduct; nucleophilicity; pre-steady-state kinetics; reactive cysteine; succinic semialdehyde dehydrogenase.