The 1′,4′-iminopyrimidine tautomer of thiamin diphosphate is poised for catalysis in asymmetric active centers on enzymes

Abstract

Thiamin diphosphate, a key coenzyme in sugar metabolism, is comprised of the thiazolium and 4-aminopyrimidine aromatic rings, but only recently has participation of the 4-aminopyrimidine moiety in catalysis gained wider acceptance. We report the use of electronic spectroscopy to identify the various tautomeric forms of the 4-aminopyrimidine ring on four thiamin diphosphate enzymes, all of which decarboxylate pyruvate: the E1 component of human pyruvate dehydrogenase complex, the E1 subunit of Escherichia coli pyruvate dehydrogenase complex, yeast pyruvate decarboxylase, and pyruvate oxidase from Lactobacillus plantarum. It is shown that, according to circular dichroism spectroscopy, both the 1,4-iminopyrimidine and the 4-aminopyrimidine tautomers coexist on the E1 component of human pyruvate dehydrogenase complex and pyruvate oxidase. Because both tautomers are seen simultaneously, these two enzymes provide excellent evidence for nonidentical active centers (asymmetry) in solution in these multimeric enzymes. Asymmetry of active centers can also be induced upon addition of acetylphosphinate, an excellent electrostatic pyruvate mimic, which participates in an enzyme-catalyzed addition to form a stable adduct, resembling the common predecarboxylation thiamin-bound intermediate, which exists in its 1,4-iminopyrimidine form. The identification of the 1,4-iminopyrimidine tautomer on four enzymes is almost certainly applicable to all thiamin diphosphate enzymes: this tautomer is the intramolecular trigger to generate the reactive ylide/carbene at the thiazolium C2 position in the first fundamental step of thiamin catalysis.1Ј,4Ј-iminothiamin diphospate ͉ 2-oxoacid decarboxylases ͉ active site asymmetry T he hypothesis that the 4Ј-aminopyrimidine (AP) ring of the coenzyme thiamin diphosphate (ThDP) (1, 2) actively participates in the reaction of enzymes that use it was suggested some years ago (3, 4). The hypothesis gained wider acceptance with the observation of two highly conserved features noted in the first structures of ThDP-dependent enzymes (5-7): (i) a distance shorter than 3.5 Å between the N4Ј atom of the AP and the C2 atom of the thiazolium ring, creating the possibility for intramolecular proton abstraction to generate the C2 carbanion/ ylide, identified by Breslow (8) as the nucleophile that initiates the sequence of reactions involving multiple ThDP-bound covalent intermediates [exemplified with yeast pyruvate decarboxylase (YPDC) (EC 4.1.1.1) in Scheme 1] and (ii) a glutamate residue within short hydrogen bonding distance of the N1Ј atom, poised to catalyze the tautomerization shown on the left side of Scheme 1. The tautomerization reaction requires three forms of the AP ring of which two are neutral, the AP and 1Ј,4Ј-iminopyrimidine (IP), but these forms must interconvert via the positively charged, N1Ј-protonated 4Ј-aminopyrimidinium ion (APH ϩ ). Our goal is to establish which form of the AP ring is present on each intermediate on the pathway in Scheme 1, and indeed in all ThDP enzymes. We here report...

Structural basis for membrane binding and catalytic activation of the peripheral membrane enzyme pyruvate oxidase from Escherichia coli

“…4A). The thiamin cofactor adopts the canonical V conformation juxtaposing the reactive C2 carbon of the thiazolium and the 4Ј-amino group of the pyrimidine ring, which acts as an intramolecular acid/base catalyst with high effective molarity and delicately balanced protonic equilibria (31)(32)(33). The isoalloxazine part of FAD is slightly bent over the N5-N10 axis (15°distortion), a structural feature that is thought to be beneficial for ET as this conformation resembles the reduced state of the flavin (27).…”

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

“…Interpretation of our results is aided by our assignment of a negative CD band centered near 320-330 nm to (i) the 4Ј-aminopyrimidine tautomer of ThDP (AP form in SI Scheme I; ref. 8) or (ii) the Michaelis complex (MC) formed with either substrate (9) or substrate analogue MAP (9,10). We have shown that on E1ec, it is difficult to observe the AP form.…”

Nuclear Magnetic Resonance Evidence for the Role of the Flexible Regions of the E1 Component of the Pyruvate Dehydrogenase Complex from Gram-negative Bacteria

“…After 45 min, samples were centrifuged for 10 min at 17,530 ϫ g, and the subcomplex was applied to the column. Circular dichroism (CD) experiments were carried out on an Applied Photophysics Chirascan spectrometer (Leatherhead, UK) at 30°C as reported earlier from our laboratories (8,10,20).…”