The role of phosphagen specificity loops in arginine kinase

Abstract

Phosphagen kinases catalyze the reversible transfer of a phosphate between ATP and guanidino substrates, a reaction that is central to cellular energy homeostasis. Members of this conserved family include creatine and arginine kinases and have similar reaction mechanisms, but they have distinct specificities for different guanidino substrates. There has not been a full structural rationalization of specificity, but two loops have been implicated repeatedly. A small domain loop is of length that complements the size of the guanidino substrate, and is located where it could mediate a lock-and-key mechanism. The second loop contacts the substrate with a valine in the methyl-substituted guanidinium of creatine, and with a glutamate in the unsubstituted arginine substrate, leading to the proposal of a discriminating hydrophobic/hydrophilic minipocket. In the present work, chimeric mutants were constructed with creatine kinase loop elements inserted into arginine kinase. Contrary to the prior rationalizations of specificity, most had measurable arginine kinase activity but no creatine kinase activity or enhanced phosphocreatine binding. Guided by structure, additional mutations were introduced in each loop, recovering arginine kinase activities as high as 15% and 64% of wild type, respectively, even though little activity would be expected in the constructs if the implicated sites had dominant roles in specificity. An atomic structure of the mismatched complex of arginine kinase with creatine and ADP indicates that specificity can also be mediated by an active site that allows substrate prealignment that is optimal for reactivity only with cognate substrates and not with close homologs that bind but do not react.

The Structure of Lombricine Kinase

“…(Such asymmetry was cited as supporting negative cooperativity (61,62), but this argument has been undercut by a glycocyamine kinase structure with both subunits in the closed substrate-bound configuration (19).) Lombricine was not available, and the concentration of the alternative substrate, taurocyamine, limited by solubility to 3ϫ K m , may not have been sufficient to obtain TSAC crystals, even though LpAK has been crystallized with a wide variety of weakly binding substrate analogs (15). 3 …”

“…Despite the different structures observed, the loop is highly conserved and identical in sequence between LK and CKs, except for UcLK residues 312-313. The equivalent of residue 312, valine in all of the CKs, but glutamate in LK, GK, and AK forms a specificity pocket that discriminates methyl-guanidino substrates (like creatine) from the other phosphagen substrates (15,16,21). Thus, the loop motions, on which UcLK sheds light, are likely critical to function.…”

“…One set (LpAK loop 59 -64) and Urechis caupo lombricine kinase (UcLK) loop [53][54][55][56][57][58] is located in the small N-terminal ␣-helical domain and facilitates binding of the phosphagen substrate through backbone hydrogen bonds to the carboxylate of the substrate. With loop length inversely correlated to substrate size, the mechanism of the small domain specificity loop has been rationalized in terms of lock-and-key steric hindrance (15,(17)(18)(19).…”

“…However, mediation of specificity is more complex than lock-and-key. In a chimeric construct with the CK specificity determinants in an LpAK background, it is possible to regain AK activity with additional mutations (15). Furthermore, a creatine-LpAK structure shows that creatine is not excluded from the active site but is imperfectly aligned with the nucleotide (15).…”

Loop Movement and Catalysis in Creatine Kinase

“…The flexible loop containing His66 has been described as a guanidino specificity loop (13,26,27). A sequence alignment for this region suggests a high level of sequence identity across the CK family and that the region is distinctly different from other guanidino kinases (Fig.…”