Journal
ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Volume 702, Issue -, Pages -Publisher
ELSEVIER SCIENCE INC
DOI: 10.1016/j.abb.2021.108819
Keywords
Enzyme; Enzyme inhibitor; Enzyme structure; Spectroscopy; Crystallography; Acetopyruvate
Categories
Funding
- Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health [P20GM1034472, P20GM103640]
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Dihydrodipicolinate synthase (DHDPS) catalyzes the initial step in the biosynthesis of L-lysine, and is a potential drug target due to its absence in mammals. Acetopyruvate is a slow-binding inhibitor of DHDPS that competes with pyruvate to form a covalent enamine complex, confirmed by spectral studies and crystal structure analysis.
Dihydrodipicolinate synthase (DHDPS) catalyzes the first step in the biosynthetic pathway for production of L-lysine in bacteria and plants. The enzyme has received interest as a potential drug target owing to the absence of the enzyme in mammals. The DHDPS reaction is the rate limiting step in lysine biosynthesis and involves the condensation of L-aspartate-beta-semialdehyde and pyruvate to form 2, 3-dihydrodipicolinate. 2, 4-oxo-pentanoic acid (acetopyruvate) is a slow-binding inhibitor of DHDPS that is competitive versus pyruvate with an initial Ki of about 20 mu M and a final inhibition constant of about 1.4 mu M. The enzyme:acetopyruvate complex displays an absorbance spectrum with a lambda(max) at 304 nm and a longer wavelength shoulder. The rate constant for formation of the complex is 86 M-1 s(-1). The enzyme forms a covalent enamine complex with the first substrate pyruvate and can be observed spectrally with a lambda(max) at 271 nm. The spectra of the enzyme in the presence of pyruvate and acetopyruvate shows the initial formation of the pyruvate enamine intermediate followed by the slower appearance of the E:acetopyruvate spectra with a rate constant of about 0.013 s(-1). The spectral studies suggest the formation of a Schiff base between acetopyruvate and K161 on enzyme that subsequently deprotonates to form a resonance stabilized anion similar to the enamine intermediate formed with pyruvate. The crystal structure of the E:acetopyruvate complex confirms the formation of the Schiff base between acetopyruvate and K161.
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