RUI: Creatine Kinase: Structure/Function Relationships of Conserved Residues by Site-Directed Mutagenesis

College Of Wooster, Wooster OH

Investigators

Abstract

9982401 Borders Creatine kinase (CK) is a key enzyme in excitable tissues, which require large energy fluxes, i.e., skeletal muscle, heart muscle, and brain. CK catalyzes the readily reversible phosphorylation of creatine by ATP to produce phosphocreatine and ADP. The major role of CK in excitable tissues is to allow the cells to accommodate rapid-ATP-producing or -ATP-depleting activities without significant fluctuation in the narrow [ATP]/[ADP] range that is necessary for optimal cell function. The proposed research would contribute to a better understanding of the mechanism of this important enzyme. The work would also contribute to a broader understanding of other fundamental areas of enzyme/protein chemistry, including the factors involved in substrate binding, catalysis and accompanying transition-state stabilization, subunit-subunit association to form oligomeric proteins, and the role of "buried" arginines in the stabilization of protein structure. CK is a member of a larger family of phosphagen kinases (PKs) that use MgATP to reversibly phosphorylate a guanidino substrate. CK is the only PK found in vertebrates, while arginine kinase (AK) is the major PK found in invertebrates. Sequence homology analysis of 25 CKs and 12 AKs has identified 71 residues that are fully conserved in all PKs, suggesting that these residues play a similar role in all guanidino kinases. Another 19 residues are fully conserved in CK and fully conserved as a different residue in AK, suggesting that these residues may play roles in differentiating substrate specificity and quaternary structure (AK is a monomer, while CK is a homodimer). This sequence homology analysis, along with the examination of the x-ray crystal structures of an AK with a bound transition-state analog complex and two different CKs, has been used to propose specific roles for the conserved residues. Site-directed mutagenesis of rabbit muscle CK will be used to examine residues involved in: 1) substrate binding; 2) catalysis; 3) transition-state stabilization; 4) monomer-monomer contacts, with the goal of forming a catalytically-active CK monomer; 5) stabilization of the tertiary structure of CK subunits, with the initial focus on two buried "structural" arginines; and 6) creatine binding, with the goal of altering the guanidino substrate specificity of the enzyme. Each mutant will be characterized by extensive kinetic analysis of the "forward" reaction (phosphocreatine formation) and, where desirable, the "reverse" reaction (creatine formation). Circular dichroic spectra and differential scanning calorimetry will be used to examine the thermal stability of each mutant, while quenching of intrinsic protein fluorescence by added nucleotide will be used to determine the nucleotide affinity and diminished transition-state stabilization of low-activity mutants. Finally, the x-ray crystal structures of selected mutants will be determined in collaboration with Dr. J. K. Mohana Rao at the Frederick Cancer Institute.