Catalytic site interactions in yeast OMP synthase.

Area of Science:

• Biochemistry
• Enzymology
• Protein Structure and Function

Background:

• Microbial OMP synthases (orotate phosphoribosyltransferase, EC 2.4.2.10) exhibit complex kinetics, including half-of-the-sites binding and structural asymmetry.
• These properties have been hypothesized to arise from an alternating site mechanism in these domain-swapped enzymes.

Purpose of the Study:

• To investigate the kinetic mechanism of yeast orotate phosphoribosyltransferase.
• To elucidate the role of specific residues in the catalytic loop and 5-phosphoribosyl-1-diphosphate (PRPP) binding motif in enzyme function and cooperativity.

Main Methods:

• Site-directed mutagenesis of conserved residues in the catalytic loop and PRPP-binding motif.
• Enzyme kinetics assays, including initial velocity studies and double reciprocal plots.
• Analysis of enzyme complementation between mutant proteins.
• Equilibrium binding studies of PRPP and orotidine 5'-monophosphate.

Main Results:

• Mutation of Lys106, a proposed intersubunit communication residue, resulted in intersecting kinetic plots and a 2-fold decrease in kcat.
• Mutants in the catalytic loop (R105G K109S H111G) and PRPP-binding motif (D131N D132N) lacked enzymatic activity and PRPP binding.
• Complementation of these mutants yielded a heterodimer with a single functional active site and intersecting initial velocity plots.
• Equilibrium binding data indicated a single class of two binding sites per dimer in wild-type and K106S enzymes, contradicting half-of-the-sites binding.

Conclusions:

• The yeast orotate phosphoribosyltransferase does not operate via a half-of-the-sites cooperativity mechanism.
• Interplay between catalytic sites is not a critical feature of the enzyme's catalytic mechanism.
• Observed parallel lines in steady-state kinetics are likely a consequence of tight substrate binding rather than an alternating site mechanism.