Generation of an Active Monomer of Rabbit Muscle Creatine Kinase by Site-Directed Mutagenesis: The Effect of Quaternary Structure on Catalysis and Stability
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- 作者：Julia M. Cox ; Caroline A. Davis ; Chikio Chan ; Michael J. Jourden ; Andrea D. Jorjorian ; Melissa J. Brym ; Mark J. Snider ; Charles L. Borders ; Jr. ; and Paul L. Edmiston
- 刊名：Biochemistry
- 出版年：2003
- 出版时间：February 25, 2003
- 年：2003
- 卷：42
- 期：7
- 页码：1863 - 1871
- 全文大小：214K
- 年卷期：v.42,no.7(February 25, 2003)
- ISSN：1520-4995
文摘
Cytosolic creatine kinase exists in native form as a dimer; however, the reasons for thisquaternary structure are unclear, given that there is no evidence of active site communication and moreprimitive guanidino kinases are monomers. Three fully conserved residues found in one-half of the dimerinterface of the rabbit muscle creatine kinase (rmCK) were selectively changed to alanine by site-directedmutagenesis. Four mutants were prepared, overexpressed, and purified: R147A, R151A, D209A, andR147A/R151A. Both the R147A and R147A/R151A were confirmed by size-exclusion chromatographyand analytical ultracentrifugation to be monomers, whereas R151A was dimeric and D209A appeared tobe an equilibrium mixture of dimers and monomers. Kinetic analysis showed that the monomeric mutants,R147A and R147A/R151A, showed substantial enzymatic activity. Substrate binding affinity by R147A/R151A was reduced approximately 10-fold, although kcat was 60% of the wild-type enzyme. Unlike theR147A/R151A, the kinetic data for the R147A mutant could not be fit to a random-order rapid-equilibriummechanism characteristic of the wild-type, but could only be fit to an ordered mechanism with creatinebinding first. Substrate binding affinities were also significantly lower for the R147A mutant, but kcat was11% that of the native enzyme. Fluorescence measurements using 1-anilinonaphthalene-8-sufonate showedthat increased amounts of hydrophobic surface area are exposed in all of the mutants, with the monomericmutants having the greatest amounts of unfolding. Thermal inactivation profiles demonstrated that proteinstability is significantly decreased in the monomeric mutants compared to wild-type. Denaturationexperiments measuring 
max of the intrinsic fluorescence as a function of guanidine hydrochlorideconcentration helped confirm the quaternary structures and indicated that the general unfolding pathwayof all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is nota prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.
max of the intrinsic fluorescence as a function of guanidine hydrochlorideconcentration helped confirm the quaternary structures and indicated that the general unfolding pathwayof all the mutants are similar to that of the wild-type. Collectively, the data show that dimerization is nota prerequisite for activity, but there is loss of structure and stability upon formation of a CK monomer.