Induced fit in guanidino kinases—comparison of substrate‐free and transition state analog structures of arginine kinase

Abstract

Arginine kinase (AK) is a member of the guanidino kinase family that plays an important role in buffering ATP concentration in cells with high and fluctuating energy demands. The AK specifically catalyzes the reversible phosphoryl transfer between ATP and arginine. We have determined the crystal structure of AK from the horseshoe crab (Limulus polyphemus) in its open (substrate-free) form. The final model has been refined at 2.35 Å with a final R of 22.3% (R free ‫ס‬ 23.7%). The structure of the open form is compared to the previously determined structure of the transition state analog complex in the closed form. Classically, the protein would be considered two domain, but dynamic domain (DynDom) analysis shows that most of the differences between the two structures can be considered as the motion between four rigid groups of nonsequential residues. ATP binds near a cluster of positively charged residues of a fixed dynamic domain. The other three dynamic domains close the active site with separate hinge rotations relative to the fixed domain. Several residues of key importance for the induced motion are conserved within the phosphagen kinase family, including creatine kinase. Substantial conformational changes are induced in different parts of the enzyme as intimate interactions are formed with both substrates. Thus, although induced fit occurs in a number of phosphoryl transfer enzymes, the conformational changes in phosphagen kinases appear to be more complicated than in prior examples.

The Structure of Lombricine Kinase

“…The protein structure is similar to that of substrate-free UcLK (cyan), except for His 178 , which has distinctly different side chain density. Although the ADP is similar to that in the LpAK-TSA complex (dark brown) (20), with mostly similar interactions, subdomain 3 is much more like substrate-free LpAK (light brown) (35). …”

“…Catalytic rates of ϳ135 s Ϫ1 (29,30) are consistent with turnover-limiting protein conformational changes (20,(31)(32)(33). Paired substratefree and transition state analog complex (TSAC) structures are available for both arginine kinase and creatine kinase; the TSAC has bound phosphagen, ADP, and a nitrate mimicking the ␥-phosphoryl in transit (21,35). Upon substrate binding there are domain reorientations up to 18°, together with the ordering and/or reconfiguring of two flexible loops over the substrates.…”

Two-domain arginine kinase from the deep-sea clam Calyptogena kaikoi—Evidence of two active domains

“…However, each domain shows a much higher degree of identity (over 80%) with the corresponding domain of other heterodont AKs. The crystal structure of the substrate-bound, closed state of Limulus AK revealed a variety of residues interacting with the substrates, arginine and ADP, as well as the key residues involved in stabilization towards large conformational changes upon substrate binding [Zhou et al, 1998;Yousef et al, 2003]. Comparison of the amino acid sequence of…”

The Putative Catalytic Bases Have, at Most, an Accessory Role in the Mechanism of Arginine Kinase

“…Several phosphagen kinase structures have become available recently, including creatine kinases from chicken muscle mitochondria (8), rabbit muscle (9), chicken brain (10), human mitochondria (ubiquitous) (11), and arginine kinase from horseshoe crab (12), all structures determined in the open, inactive configuration. The structure of arginine kinase was also determined as a transition state complex (TSAC 1 ; Mg 2ϩ -ADP, nitrate, arginine) (7) in a closed conformation with ordered active site loops.…”