The pyridoxal‐5′‐phosphate‐dependent catalytic antibody 15A9: its efficiency and stereospecificity in catalysing the exchange of the α‐protons of glycine

Abstract

IQ C-NMR has been used to follow the exchange of the K K-protons of [2-IQ C]glycine in the presence of pyridoxal-5P-phosphate and the catalytic antibody 15A9. In the presence of antibody 15A9 the 1st order exchange rates for the rapidly exchanged proton of [2-IQ C]glycine were only 25 and 150 times slower than those observed with tryptophan synthase (EC 4.2.1.20) and serine hydroxymethyltransferase (EC 2.1.2.1). The catalytic antibody increases the 1st order exchange rates of the K K-protons of [2-IQ C]glycine by at least three orders of magnitude. We propose that this increase is largely due to an entropic mechanism which results from binding the glycinepyridoxal-5P-phosphate Schiff base. The 1st and 2nd order exchange rates of the pro-2S proton have been determined but we were only able to determine the 2nd order exchange rate for the pro-2R proton of glycine. In the presence of 50 mM glycine the antibody preferentially catalyses the exchange of the pro-2S proton of glycine. The stereospecificity of the 2nd order exchange reaction was quantified and we discuss mechanisms which could account for the observed stereospecificity.z 1998 Federation of European Biochemical Societies.

From cofactor to enzymes. The molecular evolution of pyridoxal‐5′‐phosphate‐dependent enzymes

“…In the second screening step, the aldimine‐binding antibodies were screened for a catalytic effect, for example, the cleavage of the Cα‐H bond of the substrate moiety. Antibody 15A9 complied with the selection criteria and was found to catalyze the following reactions: formation of aldimine, deprotonation at Cα as reflected by β‐elimination of β‐chloroalanine ( k cat = 0.8 s –1 at 25°C), stereoselective exchange of the α‐protons of glycine,49 and transamination of PLP with hydrophobic D ‐amino acids ( k cat = 0.007 s –1 at 25°C). The screening protocol thus plausibly simulates the functional selection pressures that probably have been operative in the molecular evolution of protein‐assisted pyridoxal catalysis.…”

Identification of Functionally Important Residues in the Pyridoxal-5‘-Phosphate-Dependent Catalytic Antibody 15A9

“…As we have previously reported, antibody 15A9 catalyzes the transamination reaction of D-alanine with PLP through Antibody 15A9 in the presence of PLP catalyzes the exchange of the pro-2S proton of [2- 13 C] glycine about 6 times faster than that of the pro-2R proton (42). The preferential deprotonation of the pro-2S proton of glycine agrees with the exclusive transamination of D-amino acids, as exchange of the pro-2S proton of glycine corresponds to the deprotonation at CR of D-amino acids.…”

Catalytic antibodies and other biomimetic catalysts

“…Malthouse and co-workers have produced an antibody, 15A9, capable of catalysing the exchange of the α-hydrogens of glycine once it had formed a Schiff's base with pyridoxal 5Јphosphate. 65 The exchange of the pro-S hydrogen is accelerated an order of magnitude more than that of the pro-R hydrogen. The authors argue that the acceleration provided by the antibody is due to the trapping of the Schiff base in a position which allows maximal overlap between the α-C-H bond being cleaved and the π-orbitals of the pyridinium electron sink, rather than the involvement of an active-site base.…”