Biochemistry - The Chemical Reactions of Living Cells, Volume 2 - D. Metzler 1980

Coenzymes — special naturally occurring specialized reagents
Thiamine diphosphate
Stereochemistry of PLP-dependent enzymes

In 1966, Dunathan postulated that the bond cleaved by a PLP-dependent enzyme in the substrate amino acid must lie in a plane perpendicular to the $\pi$-System of the substrate-coenzyme imine (Fig. 8-8). This orientation minimizes the transition-state energy because it allows the maximum possible $\sigma$-$\pi$ overlap between the bond to be broken and the conjugated $\pi$-system of the cofactor imine. Furthermore, this geometry closely approaches the proposed planar quinoid intermediate, thereby minimizing molecular displacements as the Transition State is approached [47]. Figure 8-8 illustrates Three types of amino acid orientation in which the $\alpha$-hydrogen, the carboxyl group, and the side chain, respectively, are positioned optimally for bond Cleavage. For each of the shown orientations, a 180° Rotation of the amino acid yields another conformation that is also competent for the Cleavage of the specified bond.

Dunathan's concept accounts for certain Side Reactions observed in PLP-dependent Enzymes and has been supported by the experiments of Bailey and coworkers in their study of $\alpha$-dialkylamino acid aminotransferase isolated from soil Bacteria [48]. This enzyme normally catalyzes a coupled decarboxylation-Transamination half-reaction:

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FIG. 8-8. Stereochemical aspects of catalysis by PLP-dependent enzymes.

This enzyme also acts on D- and L-Alanine: specifically, while decarboxylating D-alanine, it catalyzes only the abstraction of the $\alpha$-hydrogen from L-alanine. These findings can be reasonably explained by assuming that the enzyme possesses a specific anchoring site for one alkyl group, whereas THE POSITION OF the second alkyl group can be occupied by either an $\alpha$-hydrogen or a carboxylate ion, and that the group undergoing labilization is oriented perpendicularly to the $\pi$-system:

Glycine is not a substrate for this enzyme; it follows that occupancy of the alkyl-binding site is essential for catalysis.

If Dunathan's hypothesis holds true for aminotransferases, only two viable Conformations of The amino acid substrate exist, one of which is illustrated in Fig. 8-8. In the alternative conformation, the amino acid is rotated by 180° such that the $\alpha$-hydrogen lies behind the plane of the page. Dunathan selected pyridoxamine-Pyruvate aminotransferase for his study—an enzyme related to PLP-dependent aminotransferases, yet utilizing pyridoxamine and pyridoxal as substrates¹⁾:

Using pyridoxal and L-alanine containing deuterium (²H) at the α-position as substrates, he proved the direct transfer of the amino acid α-hydrogen to the 4'-position of the resulting pyridoxamine (marked with asterisks in Fig. 8-8). It was further shown that ²H was incorporated selectively into the pro-5 position of the 4'-carbon of pyridoxamine. These results suggest that a certain protein group abstracts a proton from the α-position of the Amino Acid and transfers it across the same face of the π-system (syn-transfer), adding it to the si-face of the C = N group. Dunathan concluded that the orientation of the amino acid substrate matches the one shown in Fig. 8-8.

According to other experiments, the reduction of both the aldimine and the ketimine formed by the substrates of this enzyme with sodium borotritiide (NaB³H₄) also occurs from the si-face, which apparently indicates that this face faces the surface accessible to interactions.

Decarboxylation of amino Acids in ²Н₂О leads to the incorporation of ²Н into the pro-R position — the position originally occupied by the carboxyl group (Fig. 8-8). Cleavage of Serine by serine oxymethylase in Water containing ³Н leads to the incorporation of ³Н into the pro-S position. Stereospecific incorporation of ²Н or ³Н into the ß-position of a-ketobutyrate, obtained via ß- or у-elimination reactions, has also been observed. The conversion of serine to Tryptophan catalyzed by tryptophan synthase proceeds with retention of configuration at C-3 [49].

¹⁾ Reaction (8-26) is also catalyzed by the apoenzyme of aspartate aminotransferase, exhibiting the same direct stereospecific proton transfer as in the case of pyridoxamine:pyruvate aminotransferase [47]. It is suggested that similar results could be obtained with Pyridoxal phosphate.