Biochemical Characterization of IMP Dehydrogenase from Mycobacterium
tuberculosis
Rostirolla, D.C., de Assunção, T.M., Bizarro, C.V., Basso, L.A, Santos, D.S.
Centro de Pesquisas em Biologia Molecular e Funcional, Instituto Nacional de
Ciência e Tecnologia em Tuberculose, PUCRS, Porto Alegre, RS, Brazil.
IMP dehydrogenase (IMPDH) is the gateway to guanine nucleotides, since it
catalyses the oxidation of IMP to XMP with the conversion of NAD+ to NADH. IMPDH
from M. tuberculosis (MtIMPDH) has been predicted to be essential for mycobacterial
growth, therefore, it is proposed as a potential target to combat tuberculosis. We have
cloned and expressed the guaB2 gene product in E. coli and MtIMPDH was purified
to homogeneity. LC-MS/MS peptide mapping confirmed the identity of the
recombinant MtIMPDH protein. Apparent steady-state kinetics revealed that
MtIMPDH exhibits cooperative kinetics towards IMP and substrate inhibition is
observed when varying the concentration of NAD+. The MtIMPDH is activated by K+
and NH4+, with comparable dissociation constants, although NH4+ abolishes the
cooperativity towards IMP. In addition, the enzyme undergoes allosteric regulation,
with GTP acting as an uncompetitive inhibitor towards NAD+.The initial velocity versus
APAD indicated that the hydride transfer is not the rate-limiting step in the reaction of
MtIMPDH, since it resulted in very similar kinetic parameters for the reaction with
NAD+. Accordingly, it is hoped that the results here described may be useful to the
design of anti-TB agents and that they may contribute to our understanding of the
biology of M. tuberculosis.
Word-keys: allosteric regulation, IMP dehydrogenase, M. tuberculosis.
Supported by: CNPq.
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