Structural basis for the mechanism and substrate specificity of glycocyamine kinase, a phosphagen kinase family member.

Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg2+-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phosphoglycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicenter organisms that encounter high and variable energy turnover. GK front the mantle worm Namalycastis sp. Is heterodimeric, With two homologous polypeptide chains, alpha and beta, derived from a common pre-mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GK beta encodes a peptide that is different in sequence and is 16 amino acids longer than that encoded by the N-terminal exon of GK alpha. The crystal Structures of recombinant GK alpha beta and GK beta beta from Namalycastis sp. were determined at 2.6 and 2.4 angstrom resolution, respectively. In addition, the structure of the GK beta beta was determined at 2.3 angstrom resolution in complex with a transition state analogue. Mg2+-ADP-NO3--glycocyamine Consistent with the sequence homology, file GK subunits adopt the same overall fold its that of other phospha-en kinases of known structure (the homodimeric creatine kinase (CK) and the monomeric arginine kinase (AK)). As with CK, the GK N-termini mediate the dimer interface. In both heterodimeric and homodimeric GK forms, the conformations of the two N-termini are asymmetric, and the asymmetry is different than that reported previously for the homodimeric CKs from several organisms. The entire polypeptide chains of GK alpha beta are structurally defined, and the longer N-terminus of the beta subunit is anchored at the dimer Interface. In GK beta beta the 24 N-terminal residues of one subunit and 11 N-terminal residues of the second subunit are disordered. This observation is consistent with a proposal that the GK alpha beta amino acids involved ill the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer Interface (either solely alpha or solely beta chains) has been corrupted. Ill the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analogue, both subunits of GK undergo the same Closure motion that, clasps the transition State analogue, In contrast to the transition state analogue complexes of CK, where the corresponding transition State analogue Occupies only one subunit, which undergoes domain closure. The active site environments of the GK, CK, and AK at the bound states reveal the structural determinants Of Substrate Specificity. Despite the equivalent binding ill both active sites of the G K dimer, the conformational asymmetry of the N-termini is retained. Thus, the coupling between the structural asymmetry and negative cooperativity previously proposed for CK is not Supported ill the case of GK.