Electronic and Nuclear Magnetic Resonance Spectroscopic Features of the 1‘,4‘-Iminopyrimidine Tautomeric Form of Thiamin Diphosphate, a Novel Intermediate on Enzymes Requiring This Coenzyme

Abstract

Appropriate compounds were synthesized to create models for the 1',4'-imino tautomer of the 4'-aminopyrimidine ring of thiamin diphosphate recently found to exist on the pathway of enzymatic reactions requiring this cofactor (Jordan, F., and Nemeria, N.S. (2005) Bioorganic Chemistry, 33, 190−215). The N1-methyl-4-aminopyrimidinium compounds synthesized on treatment with a strong base produce the 1,4-imino tautomer whose UV spectrum indicates a maximum between 300−320 nm, depending on the absence or presence of a methyl group at the 4-amino nitrogen. The λ max found is in the same wavelength range as the positive circular dichroism band observed on several enzymes and showed a very strong dependence on solvent dielectric constant. To help with the 15 N chemical shift assignments, the model compounds were specifically labeled with 15 N at the amino nitrogen atom. The chemical shift of the amino nitrogen was deshielded by N1-methylation, then dramatically further deshielded by more than 100 ppm on formation of the 1,4-iminopyrimidine tautomer. Both the UV spectroscopic values and the 15 N chemical shift for the 1,4-iminopyrimidine tautomer should serve as useful guides to the assignment of enzyme-bound signals.The notion that the 4'-aminopyrimidine group of thiamin diphosphate (ThDP) undergoes tautomerization to the 1',4'-imino form (1',4'-iminoThDP) during the catalytic cycle of enzymes that utilize it has gained wider acceptance since the appearance of X-ray crystal structural data (1). The role and likelihood of tautomerization is suggested by two totally conserved structural features on all ThDP enzymes: (a) the V coenzyme conformation ensuring that the C2 thiazolium atom and the N4' atom of the 4'-aminopyrimidine ring are within less than 3.5Å from each other (2), potentially enabling the 1',4'-imino tautomer to participate in proton transfers; and (b) the presence of a glutamate within hydrogen bonding distance of the N1' atom of the 4-aminopyrimidine ring, as a potential catalyst for the tautomerization (Scheme 1, as exemplified with the reaction of yeast pyruvate decarboxylase, YPDC). As illustrated in the active site structure of YPDC in Figure 1, the residue E51 probably carries out this function (3). Chemical evidence for the importance of the 4'-aminopyrimidine moiety of ThDP in catalysis was obtained from ThDP analogues in which one or another nitrogen atom was replaced by carbon (4), while a model for activation of the ring for catalysis via tautomerization by N1-protonation was suggested from our laboratories (5-7). Notwithstanding the attractive features of this hypothesis, until recently no direct spectroscopic or structural evidence was available on any ThDP enzymes for the presence of the 1',4'-imino tautomer. In rapid-scan stopped flow experiments, mixing slow active-center variants of YPDC with pyruvate, a UV # Supported by National Institutes of Health grants GM050380 and GM062330. In these examples, the keto group of the substrate analog phosphonate forms a covalent adduct with the t...

Synthesis with good enantiomeric excess of both enantiomers of α-ketols and acetolactates by two thiamin diphosphate-dependent decarboxylases

“…This raises a significant complication in the interpretation of UV-VIS and CD data on ThDP enzymes. As we had reported, the enamine derived from aliphatic 2-oxo acids has a λ max near 295 nm [5,6], the 1',4'-imino tautomer of ThDP when enzyme-bound has a λ max between 300-310 nm with a positive CD band [19][20][21][22], and, as is here shown, acetolactate (or derivative derived from β-hydroxypyruvate), also gives rise to a positive or negative CD band with λ max of 300-302 nm (sign depending on the particular enantiomer) and a corresponding UV signal.…”

Experimental observation of thiamin diphosphate-bound intermediates on enzymes and mechanistic information derived from these observations

“…In the N1′-methylpyrimidinium the pK a of the exocyclic amine is reduced to ca. 12-12.5 [38,47], offering rationalization for the presence of conserved glutamate as a catalyst for the amino ⇌ imino tautomeric equilibration.The first evidence for a spectroscopic signature for the IP tautomer was found by Sergienko on the slow E477Q YPDC variant [45]. In a series of studies, Zhang [45] and later Baykal [47] showed that an appropriate chemical model for the 1′,4′-iminopyrimidine will give rise to a UV absorption in the 300-310 nm range.…”

“…12-12.5 [38,47], offering rationalization for the presence of conserved glutamate as a catalyst for the amino ⇌ imino tautomeric equilibration.The first evidence for a spectroscopic signature for the IP tautomer was found by Sergienko on the slow E477Q YPDC variant [45]. In a series of studies, Zhang [45] and later Baykal [47] showed that an appropriate chemical model for the 1′,4′-iminopyrimidine will give rise to a UV absorption in the 300-310 nm range. Serendipitously, the 15 N chemical shifts of the three species on the left hand side of Schemes 1 and 2, the two neutral and one positively charged forms of the 4′-aminopyrimidine are quite distinct [47] (early 15 N NMR experiments were reported on this issue by J.D.…”

“…In a series of studies, Zhang [45] and later Baykal [47] showed that an appropriate chemical model for the 1′,4′-iminopyrimidine will give rise to a UV absorption in the 300-310 nm range. Serendipitously, the 15 N chemical shifts of the three species on the left hand side of Schemes 1 and 2, the two neutral and one positively charged forms of the 4′-aminopyrimidine are quite distinct [47] (early 15 N NMR experiments were reported on this issue by J.D. Roberts and coworkers [48]).…”

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

“…The latter is unable to undergo dephosphonylation, and would presumably form a stable pre-decarboxylation analog of C2α-mandelylThDP (MThDP), the first covalent ThDP-bound intermediate in the decarboxylation of benzoylformate. In doing so, MBP might provide insight into the formation of intermediates with tetrahedral substitution at the C2α atom, including the state of ionization and tautomeric nature of the 4'-aminopyrimidine ring of ThDP on BAL, a major current focus of research of the Rutgers group with several collaborators (4)(5)(6)(8)(9)(10)(11)(12). We have now demonstrated in a number of ThDP-dependent enzymes that the 4'-aminopyrimidine ring of ThDP actively participates and plays a very crucial role in catalysis via its 1',4'-iminopyrimidine tautomeric form (4-7).…”