Pyridoxal 5'-phosphate: electrophilic catalyst extraordinaire

Abstract

Studies of nonenzymatic electrophilic catalysis of carbon deprotonation of glycine show that pyridoxal 5'-phosphate (PLP) strongly enhances the carbon acidity of alpha-amino acids, but that this is not the overriding mechanistic imperative for cofactor catalysis. Although the fully protonated PLP-glycine iminium ion adduct exhibits an extraordinary low alpha-imino carbon acidity (pK(a)=6), the more weakly acidic zwitterionic iminium ion adduct (pK(a)=17) is selected for use in enzymatic reactions. The similar alpha-imino carbon acidities of the iminium ion adducts of glycine with 5'-deoxypyridoxal and with phenylglyoxylate show that the cofactor pyridine nitrogen plays a relatively minor role in carbanion stabilization. The 5'-phosphodianion group of PLP likely plays an important role in catalysis by providing up to 12 kcal/mol of binding energy that may be utilized for transition state stabilization.

Figure 1

The mechanism for catalysis of deprotonation of glycine methyl ester by the coordinated action of the simple ketone acetone and a Brønsted base B. Electrophilic catalysis by acetone is a result of the 7-unit decrease in the pK_a of the α-amino carbon upon formation of the iminium ion adduct of glycine methyl ester with acetone [11]. The tertiary amine 3-quinuclidinone (Q) acts as a bifunctional catalyst of deprotonation of glycine methyl ester.

Figure 2

PLP strongly favors heterolytic cleavage of bonds to groups R₁ at the α-amino carbon of amino acids, by providing an “electron sink” to stabilize the negative charge that remains at this carbon after bond cleavage. PLP is used as a cofactor in enzyme-catalyzed racemization and transamination (R₁ = H), decarboxylation (R₁ = CO₂⁻) and retroaldol cleavage (R₁ = CH₂OH) reactions. The formal stabilized carbanion intermediate of these reactions is often referred to as a “quinonoid”, which emphasizes the extensive delocalization of negative charge from the α-imino carbon onto the pyridine ring of the PLP cofactor.

Figure 3

(A) Mechanism for the Claisen-type addition of glycine to DPL observed in D₂O at neutral pD [22]. This reaction is much faster than DPL-catalyzed deuterium exchange between D₂O and the α-amino protons of glycine, because the DPL-stabilized glycine carbanion (DPL=Gly⁻) reacts with a second molecule of DPL much faster than it undergoes protonation by solvent (k_add[DPL] >> k_p). (B) Condensation of the PLP-stabilized glycine carbanion with succinyl-CoA to form a β–keto acid in the reaction catalyzed by 5-aminolevulinate synthase. The β–keto acid undergoes cofactor-catalyzed decarboxylation at the enzyme active site.

Figure 4

The mechanism for two additional condensation reactions of DPL. A. At high pH there is nearly quantitative conversion of DPL and glycine to the DPL-glycine iminium ion adduct (DPL=Gly). This adduct then undergoes addition of the DPL-stabilized glycine carbanion DPL=Gly⁻ to form the novel products of addition of two equivalents of glycine to DPL [28]. B. The reaction between alanine and DPL gives a good yield of the dimeric products of addition of the α-pyridyl carbon of the DPL-stabilized alanine carbanion to a second molecule of DPL [29].

Figure 5

The carbon acid pK_as of glycine zwitterion and the iminium ion adducts that form from addition of glycine to DPL [30], acetone, and phenylglyoxylate [31]. The pK_a of 22 for the acetone-glycine iminium ion adduct is estimated with the assumption that iminium ion formation results in the same 7-unit decrease in carbon acid pK_a that we determined for glycine methyl ester [11].

Figure 6

A comparison of deprotonation of the PLP-alanine iminium ion adduct catalyzed by amino acid racemases and transaminases. The pyridine nitrogen of PLP is deprotonated by the strongly basic guanidine side-chain of Arg-219 at the active site of alanine racemase. The PLP-stabilized alanine carbanion is proposed to show minimal delocalization of negative charge onto the pyridine ring nitrogen. This favors charge localization at the α-imino carbon, which is then reprotonated by the racemase. The pyridine nitrogen of PLP is protonated by the carboxylic acid side-chains at the active sites of D-amino acid aminotransferase and alanine glyoxylate aminotransferase. This promotes delocalization of negative charge from the α-imino carbon of the carbanion intermediate onto the pyridine ring nitrogen. This will increase the negative charge density at the α-pyridyl carbon and protonation of this carbon results in the 1,3-proton shift catalyzed by transaminases.