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EXTRA DOMAIN KEEPS ENZYME IN THE FOLD
kathryn{at}biologists.com
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Every day, our bodies consume kilograms of ATP, which drives almost all our metabolic processes from opening our eyes in the morning, to keeping our brains functioning every moment of our lives. But making sure that a cell has enough ATP ready for the moment when it leaps into action is the job of a group of enzymes known as the phosphagen kinases, such as creatine kinase and arginine kinase. When energy demands are low they effectively store energy by transferring the terminal phosphate group from ATP to either creatine or arginine to form creatine phosphate and arginine phosphate. But when a cell needs a sudden burst of energy, the kinases retrieve the phosphate group, restoring it to an ADP molecule to produce ATP. Most arginine kinases function as a single protein molecule, but some invertebrates evolved novel forms of the protein, with two copies of the arginine kinase enzyme linked together in a single polypeptide. Ross Ellington explains that having two copies of the enzyme makes understanding the catalytic mechanism of the enzyme an intriguing problem. Working with Deanne Compaan, he began looking at the doubled-up enzyme produced by razor clams to find out how both domains function when linked together in a single enzyme (p. 1545).
Compaan and Ellington set about cloning the long enzyme. Ellington explains that the razor clam foot muscle is a `burst type muscle', so the muscle has high levels of the enzyme and the associated mRNA, `which makes our task easier' adds Ellington. Within a matter of weeks, the team had cloned the enzyme and when he looked at the enzyme's sequence, both domains had all of the amino acids that are essential for them to catalyse the energy storing reaction. But then the team hit a snag; they could make large amounts of the protein in E. coli, but only the full length enzyme behaved like a properly folded protein. When they tried expressing each domain separately, they only found unfolded protein in the E. coli cells. Fortunately Ellington had experience of rescuing denatured proteins, and after successfully refolding both domains, Campaan and Ellington began testing the enzymes' kinetics.
Sure enough, the full length enzyme, equipped with both domains, catalysed the reaction between arginine phosphate and ADP to produce ATP, and did it at twice the rate of a monomeric arginine kinase from the horseshoe crab. But when they looked at the individual domains, the team was in for a shock; the first domain didn't function at all! However, when Ellington began looking at the second domain he says that `everything fell into place'; the second domain alone accounted for all of the full-length enzyme's activity.
Why has the enzyme retained a domain that doesn't seem to be doing much, after all it costs the mollusc twice as much effort to synthesise the larger enzyme, so the extra domains must be doing something important? Ellington suspects that the extra domain comes into its own before the enzyme sees arginine phosphate or ADP; he thinks that it behaves as a chaperone, helping the full-length enzyme to fold correctly, producing a highly active enzyme, despite a `rather cumbersome evolutionary accident'.
References
Compaan, D. M. and Ellington, W. R. (2003).
Functional consequences of a gene duplication and fusion event in an arginine
kinase. J. Exp. Biol.
206,1545
-1556.
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