Comparative & Evolutionary Biochemistry; Structure, Function & Evolution of Phosphagen Kinases

 

Overview

     Phosphagen kinases are a family of phosphoryl transfer enzymes that are typically expressed in cells displaying high and variable rates of ATP turnover (muscle fibers, neurons, spermatozoa, photoreceptors, transport epithelial cells). By far the most widely distributed phosphagen kinases are arginine kinase (AK) and creatine kinase (CK) which catalyze the following reactions:

AK: Arginine-P + MgADP + H ⁺ Arginine + MgATP

CK: Creatine-P + MgADP + H ⁺ Creatine + MgATP

     The AK and CK reactions function as temporal and spatial ATP buffers, maintaining the chemical potential for ATP hydrolysis at optimal levels to mitigate temporal and spatial mismatches of ATP supply and demand.

     AK is ancient; it is present in key protozoan groups and is widespread throughout the metazoa but is lacking in the craniates (hagfish and vertebrates). CK is present in a variety of invertebrates and is the exclusive phosphagen kinase in the craniates. CK likely evolved from an AK-like ancestor via a gene duplication event followed by divergence. Subsequently, CK underwent a series of duplication and divergence events each resulting in distinct CK genes that code for proteins targeted to different intracellular compartments- cytoplasm, mitochondrion and flagellum. We have recently shown that the bulk of these CK divergence events occurred at the dawn of the radiation of multi-cellular animals. The Ellington lab is pursuing a number of fundamental questions about phosphagen kinases:

 

1. What were the early physiological roles of AK and CK? . 
    
2. When did the original CK gene emerge? . 
    
3. What was the sequence of events for the evolution of the various CK genes? And . 
    
4. What were the driving forces that led to the divergence and formation of isoforms of CK, each targeted to different intracellular compartments. . 
    

Current efforts focus on lower invertebrates (sponges and cnidarians) as well as protozoans from both the anterokont (anterior cilium) and opisthokont (posterior cilium) lineages.

Major Projects Underway

1. Evolution of the phosphagen kinase enzyme family (in collaboration with the T. Suzuki group, Kochi University). 
    
2. Evolution, structural correlates and catalytic implications of quaternary structure in phosphagen kinases. 
    
3. Evolution and functional impact of intracellular targeting in creatine kinases. 
    
4. X-ray crystallographic studies of arginine kinase focusing on mechanisms of catalysis and substrate specificity ( in collaboration with the M. Chapman group, Florida State University). 
    
5. Studies of creatine biosynthesis and transport in lower chordate and invertebrate systems.
    

Major Techniques Used in the Ellington Lab

1. Contemporary molecular biology procedures including genomic DNA PCR, RTPCR, cDNA library construction and expression and site directed mutagenesis of recombinant protein.
    
2. Protein purification and physico-chemical and catalytic characterization; steady state enzyme kinetics.
    
3. Bioinformatics procedures including sequence and phylogeny analyses and homology modeling of 3-D atomic structures.
    
4. Enzyme-linked spectrophotometric and flourimetric assays of metabolite levels in cells and tissues.
    
5. Immunofluorescence light microscopy and immunogold TEM localization of enzymes in various cells and cellular compartments.
    

Phosphagen Kinase and Phosphagen Related Papers From the Ellington Lab (2000 on)

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