%0 Journal Article
%A Czekster, Clarissa
M.
%A Blanchard, John S.
%D 2012
%T One Substrate, Five Products:
Reactions Catalyzed
by the Dihydroneopterin Aldolase from Mycobacterium
tuberculosis
%U https://acs.figshare.com/articles/journal_contribution/One_Substrate_Five_Products_Reactions_Catalyzed_by_the_Dihydroneopterin_Aldolase_from_Mycobacterium_tuberculosis/2463937
%R 10.1021/ja308350f.s001
%2 https://acs.figshare.com/ndownloader/files/4106626
%K aldolase reaction
%K tetrahydrofolate biosynthesis
%K enzyme
%K substrate
%K carbon transfer reaction
%K MtDHNA
%K DHNA
%K Mycobacterium tuberculosisTetrahydrofolate cofactors
%K proton inventory studies
%X Tetrahydrofolate cofactors are required for one carbon
transfer
reaction involved in the synthesis of purines, amino acids, and thymidine.
Inhibition of tetrahydrofolate biosynthesis is a powerful therapeutic
strategy in the treatment of several diseases, and the possibility
of using antifolates to inhibit enzymes from Mycobacterium
tuberculosis has been explored. This work focuses
on the study of the first enzyme in tetrahydrofolate biosynthesis
that is unique to bacteria, dihydroneopterin aldolase (MtDHNA). This enzyme requires no metals or cofactors and does not form
a protein-mediated Schiff base with the substrate, unlike most aldolases.
Here, we were able to demonstrate that the reaction catalyzed by MtDHNA generates three different pterin products, one of
which is not produced by other wild-type DHNAs. The enzyme–substrate
complex partitions 51% in the first turnover to form the aldolase
products, 24% to the epimerase product and 25% to the oxygenase products.
The aldolase reaction is strongly pH dependent, and apparent pKa values were obtained for the first time for
this class of enzyme. Furthermore, chemistry is rate limiting for
the aldolase reaction, and the analysis of solvent kinetic isotope
effects in steady-state and pre-steady-state conditions, combined
with proton inventory studies, revealed that two protons and a likely
solvent contribution are involved in formation and breakage of a common
intermediate. This study provides information about the plasticity
required from a catalyst that possesses high substrate specificity
while being capable of utilizing two distinct epimers with the same
efficiency to generate five distinct products.
%I ACS Publications