pubs.acs.org/Biochemistry Published on Web 11/10/2010 r 2010 American Chemical Society 10818 Biochemistry 2010, 49, 10818–10830 DOI: 10.1021/bi101061m NMR Studies of the Stability, Protonation States, and Tautomerism of 13 C- and 15 N-Labeled Aldimines of the Coenzyme Pyridoxal 5 0 -Phosphate in Water † Monique Chan-Huot, ‡ Shasad Sharif, ‡ Peter M. Tolstoy, ‡ Michael D. Toney, § and Hans-Heinrich Limbach* ,‡ ‡ Institut f € ur Chemie und Biochemie, Freie Universit € at Berlin, Takustrasse 3, D-14195 Berlin, Germany, and § Department of Chemistry, University of California-Davis, Davis, California 95616, United States Received July 2, 2010; Revised Manuscript Received November 9, 2010 ABSTRACT: We have measured the pH-dependent 1 H, 13 C, and 15 N NMR spectra of pyridoxal 5 0 -phosphate ( 13 C 2 -PLP) mixed with equal amounts of either doubly 15 N-labeled diaminopropane, 15 N R -labeled L-lysine, or 15 N ε -labeled L-lysine as model systems for various intermediates of the transimination reaction in PLP- dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their 13 C and 15 N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with L-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the R-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK a values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction. Vitamin B 6 or pyridoxal 5 0 -phosphate (1, PLP) is a cofactor of many enzymes responsible for a wide variety of amino acid transformations such as racemization, transamination, and de- carboxylation among others. The different functional groups of PLP enable a surprisingly large number of chemical states, protonation states, and tautomeric states. In the active sites of enzymes, it forms a Schiff base generally termed the “internal aldimine” with the ε-group of a lysine residue (Scheme 1). When an amino acid substrate enters the active site, the internal aldimine is converted to an “external aldimine” (Scheme 1b) in which the R-amino group of the incoming amino acid forms the Schiff base (1). This transimination is common to all PLP- dependent enzymes and has been studied by a number of authors (2-10). It has been argued that the transimination requires a positive charge on the imino nitrogen of the Schiff base which is assisted by protonation of the pyridine ring. The latter should increase the equilibrium constant of the keto-enol tautomerism between an enolimine form and a ketoamine form as illustrated in Scheme 1a (11, 12). Indeed, a coupling between the two OHN hydrogen bonds has been confirmed recently by a combination of liquid- and solid-state 15 N NMR (13-18) of model Schiff bases. Also, microsolvation favors the ketoamine form (13). In this regard, alanine racemase represents a puzzle as X-ray studies suggest that the pyridine ring of the internal aldimine of PLP is not protonated (19). Therefore, microsolvation remains here the major explanation for the activation of PLP in alanine racemase, as supported recently by theoretical calculations (20). The study of the tautomerism of PLP aldimines in water where microsolva- tion is maximized is, therefore, of special interest. For the transimination reaction itself, two different hypotheses have been discussed. As illustrated in Scheme 1b, transimination could proceed via a geminal diamine formed by a direct addition of the amino group of the incoming amino acid to the internal aldimine. This pathway requires a base which accepts at least temporarily the proton of the ammonium group of the incoming amino acid. There are several reports in the literature which provide evidence for the geminal diamine pathway. An intermediate UV absorption around 340 nm was assigned to a singly protonated geminal diamine (5-7). Recently, a geminal diamine formed by an internal lysine and an external glutamate could be trapped and observed by X-ray crystallography (21), but its protonation states remained unknown; interestingly, the solid was found also to absorb light around 340 nm. The transimination could also occur via the hydrolysis of the internal Schiff base involving a carbinolamine as intermediate (not shown) and the formation of the aldehyde or hydrate form of PLP, which are stabilized under acidic conditions (22). In a second step, the external aldimine may be formed with the sub- strate as illustrated in Scheme 1b (23, 24). This pathway involves a certain number of water molecules in the active site as well as stabilization of free PLP. To the present, it is not experimentally defined which pathway is realized and how it proceeds. However, it seemed to us that in order to obtain knowledge about the mechanism of the transimination the study of the interplay between chemical stability, protonation states, and tautomeric states under microsolvation with water molecules could be useful. † This work has been supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie, Frankfurt. *Corresponding author. Tel: þ49 3083855375. Fax: þ49 3083855310. E-mail: limbach@chemie.fu-berlin.de.