Gary S. Bild

Introducing strong metal-binding sites onto surfaces of proteins for facile and efficient metal-affinity purifications

Abstract Recombinant DNA methodologies are used to introduce strongly binding, metal-chelating sites onto the surfaces of bovine and porcine somatotropin in such a way that biological activity and protein stability are maintained. Binding to immobilized metals is sufficiently strong and selective that pure variant proteins are efficiently recovered from concentrated or dilute crude cell lysates in one or two purification steps. Chemically robust immobilized metal-affinity matrices are tolerant to repeated use and frequent cleaning and possess high protein-binding capacities. In addition to the preparative uses, immobilized metal-affinity chromatography is a sensitive tool for probing the structures of somatotropin surfaces near metal-binding residues. General principles underlying metal-protein interactions, immobilized metal-affinity chromatography, and variant design are discussed and illustrated.

Detection of β-carbanion formation during kynurenine hydrolysis catalyzed by Pseudomonas marginalis kynureninase

2-Amino-4-hydroxy-4-phenylbutyric acid has been shown to be formed during the Pseudomonas marginalis kynureninase-catalyzed hydrolysis of kynurenine in the presence of benzaldehyde and pyridoxal phosphate. The formation of 2-amino-4-hydroxy-4-phenylbutyric acid is the first demonstration, to our knowledge, of the controlled trapping of an amino acid beta-carbanion generated either chemically or enzymatically, and is perhaps the best empirical evidence to date that enzyme mechanisms can proceed through a beta-carbanionic intermediate. The lifetime of the beta-carbanionic alanyl intermediate generated by kynureninase is of sufficient duration to allow reaction with benzaldehyde. Other aromatic, but no aliphatic, aldehydes will undergo electrophilic addition with kynureninase-generated beta-carbanionic alanyl intermediates to form the corresponding amino acid.

Crystallization of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli

Crystals of 5-enolpyruvylshikimate 3-phosphate synthase from Escherichia coli have been grown out of ammonium sulfate by the hanging drop method of vapor diffusion. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with a = 124 A and c = 381 A, and diffract to 3.8 A resolution.

A Method for the Synthesis of [1]-Kynurenine

Abstract A new method is described for the synthesis of [1]-labelled L-kynurenine from [1]-L-tryptophan, using extracts of tryptophan-adapted cells of Pseudomonas marginalis. It is based on the selective, rapid inactivation of kynureninase by a newly discovered inhibitor of this enzyme, 3-chloro-L-alanine. The yield of [1]-kynurenine produced in this manner is 76% theoretical.