Specific host-guest interactions in a protein-based artificial transaminase.
Artificial enzymes can be created by covalent attachment of a catalytic active group to a protein scaffold. Recently, we assembled an artificial transaminase by conjugation of intestinal fatty acid binding protein (IFABP) with a pyridoxamine derivative via a disulfide bond; the resulting construct catalyzed a transamination reaction 200-fold faster than free pyridoxamine. To identify the origin of this increased catalytic efficiency computer modeling was first used to identify two putative residues, Y14 and R126, that were in close proximity to the gamma-carboxylate group of the substrate, alpha-ketoglutartate. These positions were mutated to phenylalanine and methionine, respectively, and used to prepare semisynthetic transaminases by conjugation to pyridoxamine (Px) or an N-methylated derivative (MPx). Kinetic analysis of the resulting constructs showed that the R126M mutation reduced 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 pyridoxamine catalyzes a transamination reaction with a k(cat)' value of 1.1h(-1) which is the fastest value for k(cat) we have thus far obtained and is 34-fold greater than that for the free cofactor in the absence of the protein.
Häring
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Department of Chemistry, University of Minnesota, Minneapolis, MN 55455, USA.
Distefano
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MD
D019943:Amino Acid Substitution; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D003198:Computer Simulation; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D006801:Humans; D007422:Intestines / Q000737:chemistry; D007700:Kinetics; D008958:Models, Molecular; D018716:Molecular Mimicry; D016297:Mutagenesis, Site-Directed; D009363:Neoplasm Proteins; D011485:Protein Binding; D011733:Pyridoxamine / Q000737:chemistry*; D013379:Substrate Specificity; D000637:Transaminases / Q000138:chemical synthesis* / Q000737:chemistry; D025521:Tumor Suppressor Proteins
D002352:Carrier Proteins; C107608:FABP7 protein, human; D000071438:Fatty Acid-Binding Protein 7; D050556:Fatty Acid-Binding Proteins; D009363:Neoplasm Proteins; D025521:Tumor Suppressor Proteins; D011733:Pyridoxamine; D000637:Transaminases