Specific host-guest interactions in a protein-based artificial transaminase

Artificial enzymes can be created by covalent attachment of a catalytic act ive group to a protein scaffold. Recently, we assembled an artificial trans aminase by conjugation of intestinal fatty acid binding protein (IFABP) wit h a pyridoxamine derivative via a disulfide bond; the resulting construct c atalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer mode ling was first used to identify two putative residues, Y14 and R126, that w ere in close proximity to the gamma -carboxylate group of the substrate, al pha -ketoglutartate. These positions were mutated to phenylalanine and meth ionine, respectively, and used to prepare semisynthetic transaminases by co njugation to pyridoxamine (Px) or an N-methylated derivative (MPx). Kinetic analysis of the resulting constructs showed that the R126M mutation reduce d substrate affinity 3- to 6-fold while the additional Y14F mutation had a negligible effect. These results are consistent with a model for substrate recognition that involves an electrostatic interaction between the cationic guanidinium group of R126 and the anionic carboxylate from the substrate. Interestingly, one of the conjugates that contains an N-methylated pyridoxa mine catalyzes a transamination reaction with a k(cat)' value of 1.1 h(-1) which is the fastest value for k(cat) we have thus far obtained and is 34-f old greater than that for the free cofactor in the absence of the protein. (C) 2001 Elsevier Science Ltd. All rights reserved.