Purification and characterization of yeast anthranilate phosphoribosyltransferase

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Abstract
Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of Mr= 83000 ± 4000 (s20,w= 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even at low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex fromation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady‐state‐kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme‐substrate complex, with KANTm= 1.6 μM, and KPRib‐PPm= 22.4 μM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylantharnilate isomerase from Escherichia coli: KPRAm=5μM, Kcat= 40 s−1.