The long-term goal of this program is elucidate the structure- function correlation for two allosteric enzymes - rabbit muscle pyruvate kinase (PK) and phosphofructokinase (PFK). Towards that goal in the PK system, 4 specific projects are proposed. (1) Since steady-state kinetics is the standard approach to study enzymes, hence, all the physicial measurements in this study are linked to steady-state kinetics. The basic mechanism of enzyme reaction for PK is still not definitely established, hence, the kinetic mechanism will be determined by stead-state kinetics and ligand equilibrium binding studies as a function of temperature and pH. This should provide the prerequisite information required for establishing a model independent procedure to extract useful thermodynamic information from steady-state kinetic data. (2) Since steady-state kinetic parameters are usually composites of different rate constants, hence, the rate constants of the elementary steps involved in PK activity will be determined by rapid kinetics. Establishment of this procedure and the information obtained enables one to propose (3) a detail study on the kidney isozyme to pinpoint the specific step altered by structural changes generated by natural mutations represented by PK isozymes. (4) Since PK isomerization is important in enzyme regulation, the global structural changes induced by ligands will be monitored by tritium exchange. Further development of this procedure coudl lead to identification of peptides responsive to allosteric changes. For the PFK system, it is now established that subunit interaction is linked to enzyme regulation, hence, it is proposed to determine: (1) The mechanism of subunit assembly. (2) The thermodynamic linkages between ligand binding and subunit assembly. (3) The structural difference between the active and inactive tetrameric form. (4) The interaction of PFK and other glycolytic enzymes with F-actin. The first 3 projects provide the fundamental knowledge upon which a quantitataive mechanism of enzyme regulation can be established. The last project represents the exploration of a higher level of complexity in the regulation of glycolytic metabolism.
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