4 research outputs found
Determination of Pre-Steady-State Rate Constants on the Escherichia coli Pyruvate Dehydrogenase Complex Reveals That Loop Movement Controls the Rate-Limiting Step
Spectroscopic identification and characterization of
covalent and
noncovalent intermediates on large enzyme complexes is an exciting
and challenging area of modern enzymology. The Escherichia
coli pyruvate dehydrogenase multienzyme complex (PDHc),
consisting of multiple copies of enzymic components and coenzymes,
performs the oxidative decarboxylation of pyruvate to acetyl-CoA and
is central to carbon metabolism linking glycolysis to the Krebs cycle.
On the basis of earlier studies, we hypothesized that the dynamic
regions of the E1p component, which undergo a disorder–order
transition upon substrate binding to thiamin diphosphate (ThDP), play
a critical role in modulation of the catalytic cycle of PDHc. To test
our hypothesis, we kinetically characterized ThDP-bound covalent intermediates
on the E1p component, and the lipoamide-bound covalent intermediate
on the E2p component in PDHc and in its variants with disrupted active-site
loops. Our results suggest that formation of the first covalent predecarboxylation
intermediate, C2α-lactylthiamin diphosphate (LThDP), is rate
limiting for the series of steps culminating in acetyl-CoA formation.
Substitutions in the active center loops produced variants with up
to 900-fold lower rates of formation of the LThDP, demonstrating that
these perturbations directly affected covalent catalysis. This rate
was rescued by up to 5-fold upon assembly to PDHc of the E401K variant.
The E1p loop dynamics control covalent catalysis with ThDP and are
modulated by PDHc assembly, presumably by selection of catalytically
competent loop conformations. This mechanism could be a general feature
of 2-oxoacid dehydrogenase complexes because such interfacial dynamic
regions are highly conserved
Bifunctionality of the Thiamin Diphosphate Cofactor: Assignment of Tautomeric/Ionization States of the 4′-Aminopyrimidine Ring When Various Intermediates Occupy the Active Sites during the Catalysis of Yeast Pyruvate Decarboxylase
Thiamin diphosphate (ThDP) dependent enzymes perform
crucial C–C
bond forming and breaking reactions in sugar and amino acid metabolism
and in biosynthetic pathways via a sequence of ThDP-bound covalent
intermediates. A member of this superfamily, yeast pyruvate decarboxylase
(YPDC) carries out the nonoxidative decarboxylation of pyruvate and
is mechanistically a simpler ThDP enzyme. YPDC variants created by
substitution at the active center (D28A, E51X, and E477Q) and on the
substrate activation pathway (E91D and C221E) display varying activity,
suggesting that they stabilize different covalent intermediates. To
test the role of both rings of ThDP in YPDC catalysis (the 4′-aminopyrimidine
as acid–base, and thiazolium as electrophilic covalent catalyst),
we applied a combination of steady state and time-resolved circular
dichroism experiments (assessing the state of ionization and tautomerization
of enzyme-bound ThDP-related intermediates), and chemical quench of
enzymatic reaction mixtures followed by NMR characterization of the
ThDP-bound intermediates released from YPDC (assessing occupancy of
active centers by these intermediates and rate-limiting steps). Results
suggest the following: (1) Pyruvate and analogs induce active site
asymmetry in YPDC and variants. (2) The rare 1′,4′-iminopyrimidine
ThDP tautomer participates in formation of ThDP-bound intermediates.
(3) Propionylphosphinate also binds at the regulatory site and its
binding is reflected by catalytic events at the active site 20 Ă…
away. (4) YPDC stabilizes an electrostatic model for the 4′-aminopyrimidinium
ionization state, an important contribution of the protein to catalysis.
The combination of tools used provides time-resolved details about
individual events during ThDP catalysis; the methods are transferable
to other ThDP superfamily members
Glyoxylate Carboligase: A Unique Thiamin Diphosphate-Dependent Enzyme That Can Cycle between the 4′-Aminopyrimidinium and 1′,4′-Iminopyrimidine Tautomeric Forms in the Absence of the Conserved Glutamate
Glyoxylate carboligase (GCL) is a thiamin diphosphate
(ThDP)-dependent
enzyme, which catalyzes the decarboxylation of glyoxylate and ligation
to a second molecule of glyoxylate to form tartronate semialdehyde
(TSA). This enzyme is unique among ThDP enzymes in that it lacks a
conserved glutamate near the N1′ atom of ThDP (replaced by
Val51) or any other potential acid–base side chains near ThDP.
The V51D substitution shifts the pH optimum to 6.0–6.2 (p<i>K</i><sub>a</sub> of 6.2) for TSA formation from pH 7.0–7.7
in wild-type GCL. This p<i>K</i><sub>a</sub> is similar
to the p<i>K</i><sub>a</sub> of 6.1 for the 1′,4′-iminopyrimidine
(IP)–4′-aminopyrimidinium (APH<sup>+</sup>) protonic
equilibrium, suggesting that the same groups control both ThDP protonation
and TSA formation. The key covalent ThDP-bound intermediates were
identified on V51D GCL by a combination of steady-state and stopped-flow
circular dichroism methods, yielding rate constants for their formation
and decomposition. It was demonstrated that active center variants
with substitution at I393 could synthesize (<i>S</i>)-acetolactate
from pyruvate solely, and acetylglycolate derived from pyruvate as
the acetyl donor and glyoxylate as the acceptor, implying that this
substitutent favored pyruvate as the donor in carboligase reactions.
Consistent with these observations, the I393A GLC variants could stabilize
the predecarboxylation intermediate analogues derived from acetylphosphinate,
propionylphosphinate, and methyl acetylphosphonate in their IP tautomeric
forms notwithstanding the absence of the conserved glutamate. The
role of the residue at the position occupied typically by the conserved
Glu controls the pH dependence of kinetic parameters, while the entire
reaction sequence could be catalyzed by ThDP itself, once the APH<sup>+</sup> form is accessible
Lipoamide Channel-Binding Sulfonamides Selectively Inhibit Mycobacterial Lipoamide Dehydrogenase
Tuberculosis remains a global health
emergency that calls for treatment
regimens directed at new targets. Here we explored lipoamide dehydrogenase
(Lpd), a metabolic and detoxifying enzyme in <i>Mycobacterium
tuberculosis</i> (Mtb) whose deletion drastically impairs Mtb’s
ability to establish infection in the mouse. Upon screening more than
1.6 million compounds, we identified <i>N</i>-methylpyridine
3-sulfonamides as potent and species-selective inhibitors of Mtb Lpd
affording >1000-fold selectivity versus the human homologue. The
sulfonamides
demonstrated low nanomolar affinity and bound at the lipoamide channel
in an Lpd–inhibitor cocrystal. Their selectivity could be attributed,
at least partially, to hydrogen bonding of the sulfonamide amide oxygen
with the species variant Arg93 in the lipoamide channel. Although
potent and selective, the sulfonamides did not enter mycobacteria,
as determined by their inability to accumulate in Mtb to effective
levels or to produce changes in intracellular metabolites. This work
demonstrates that high potency and selectivity can be achieved at
the lipoamide-binding site of Mtb Lpd, a site different from the NAD<sup>+</sup>/NADH pocket targeted by previously reported species-selective
triazaspirodimethoxybenzoyl inhibitors