Aldimine from pyridoxal phosphate

Structures of catalytic intermediates in pyridoxal-phosphate-dependent reactions. The initial aldimine intermediate resulting from Schiff s base formation between the coenzyme and the a-amino group of an amino acid (a). This aldimine is converted to the resonance-stabilized... 

In the absence of the amino acid substrate, pyridoxal phosphate is bound to enzymes by the formation of a Schiff base to the -amino group of a lysine residue at the active site. As shown in Figure 9.2, the first reaction between the substrate and the coenzyme is transfer of the aldimine linkage from the e-amino group of the lysine residue to the a-amino group of the substrate. 

The (I( )-l-amino-2-propanol linker is known to be derived from threonine. In S. enterica, CobD was found to be an enzyme with L-threonine 0-3-phosphate decarboxylase activity, which generates (/f)-l-amino-2-propa-nol phosphate. The enzyme is a pyridoxal phosphate requiring enzyme and the structure of the protein has been determined by X-ray crystallography (Figure 28). The structure of CobD was found to be highly similar to the aspartate aminotransferase family of enzymes. Structures of CobD with substrate and product bound have allowed a detailed mechanism for the enzyme to be proposed, whereby the external aldimine is directed toward decarboxylation rather than aminotransfer. Threonine phosphate, itself, is synthesized from L-threonine by the action of a kinase, which is encoded by pduX The pduX is housed within the propanediol utilization operon rather than the cobalamin biosynthetic operon for reasons that are not clear. 

Figure 11.13 Reactions at a-carbon of a-amino acids catalyzed by pyridoxal enzymes All three substituents at C are subject to labilization in the three types of a-carbon reactions. The hydrogen is labilized in recemization reactions, the amino group is labUized in the transamination and the carboxyl group is labilized in decarboxylation. a-Amino acid condenses with pyridoxal phosphate to yield pyridoxylidene imino acid (an aldimine). The common intermediate, aldimine and distinct ketimines leading to the production of oxo-acid (in transamination), amino acid (in racemization) and amine (in decarboxylation) are shown. The catalytic acid (H-A-) and base (-B ) are symbolic both can be from the same residue such as Lys258 in aspartate aminotransferase.

The /3 decarboxylation of aspartate (equation 7) proceeds by elimination of a /3-carbanionic intermediate like that in figure 10.4c/ from the ketimine, analogous to the intermediate produced by loss of the a proton from the aldimine of aspartate with pyridoxal-5 -phosphate. 

Rudnick and Abeles purified proline racemase to 95% homogeneity from Clostridium sticklandii, and characterized it 92. The enzyme is composed of two identical subunits with a molecular weight of about 38000, and is independent of any cofactors or metals. Most amino acid racemases require pyridoxal 5 -phosphate, which labilizes the bond between the a-hydrogen and the chiral center by aldimine formation with the a-amino group of the substrate. However, PLP is not involved in the reaction of proline racemase acting on an a-imino acid. The enzyme also acts on 2-hydroxy-L-proline and 2-allo-hydroxy-D-proline although slowly they are epimer-ized at a rate of 2 and 5% of the rate of L-proline racemization, respectively. L-Proline and D-proline showed Km values of 2.9 and 2.5 mti, respectively1119. ... 

As in problem 15, an external aldimine (protonated Schiff base) is formed between serine and pyridoxal-5 -phosphate, and the serine a-hydrogen is removed. The a-hy-drogen then can be reattached from either of two faces to give the Schiff base of either D-serine or L-serine (see below). The equilibrium constant for the reaction will be one. 

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