Martin Kiefer

Kinetic and spectroscopic characterization of native and metal-substituted β-lactamase from Aeromonas hydrophila AE036

Two metal ion binding sites are conserved in metallo‐β‐lactamase from Aeromonas hydrophila. The ligands of a first zinc ion bound with picomolar dissociation constant were identified by EXAFS spectroscopy as one Cys, two His and one additional N/O donor. Sulfur‐to‐metal charge transfer bands are observed for all mono‐ and di‐metal species substituted with Cu(II) or Co(II) due to ligation of the single conserved cysteine residue. Binding of a second metal ion results in non‐competitive inhibition which might be explained by an alternative kinetic mechanism. A possible partition of metal ions between the two binding sites is discussed.

Transient kinetics of ligand binding and role of the C-terminus in the dUTPase from equine infectious anemia virus.

Transient kinetics of the equine infectious anemia virus deoxyuridine 5′‐triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one‐step mechanism for substrate binding. A C‐terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C‐terminus is not a prerequisite for substrate binding, but indispensable for catalysis.

The influence of pH on the substrate specificity and stereoselectivity of alcohol dehydrogenase from horse liver.

Alcohol dehydrogenase from horse liver (HLADH) catalyzes the reversible oxidation of primary and secondary alcohols. The substrate specificity of the EE isozyme is very broad and has been characterized in depth (for review see Jones and Beck, 1976).