Functional non‐identity of creatine kinase subunits of rabbit skeletal muscle

Nevinsky, G.A.; Ankilova, V.N.; Lavrik, Olga I.; Mkrtchyan, Z.S.; Nersesova, L. S.; Akopyan, J.I.

doi:10.1016/0014-5793(82)81065-x

Cited by 11 publications

(3 citation statements)

References 7 publications

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“…The relationship between the N-termini conformational asymmetry to the observed subunit binding asymmetry are unknown, although it was recently proposed that the N-terminal regions play a role in active site communication and catalytic regulation (17). The structural asymmetry supports the view that the CK subunits exhibit negative cooperativity (22-25), whereas other chemical modification experiments and data from deuterium exchange experiments contradict the notion of negative cooperativity in CK (26, 27). The two distinct alternative spliced GK N-termini imply an inherently asymmetric structure, which is confirmed by the crystal structures of both the homodimeric GKββ and the heterodimeric GKαβ reported here.…”

supporting

confidence: 72%

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

et al. 2010

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Glycocyamine kinase (GK), a member of the phosphagen kinase family, catalyzes the Mg2+-dependent reversible phosphoryl group transfer of the N-phosphoryl group of phospho glycocyamine to ADP to yield glycocyamine and ATP. This reaction helps to maintain the energy homeostasis of the cell in some multicelullar organisms that encounter high and variable energy turnover. GK from the marine worm Namalycastis sp. is heterodimeric, with two homologous polypeptide chains, α and β, derived from a common pre mRNA by mutually exclusive N-terminal alternative exons. The N-terminal exon of GKβ encodes a peptide that is different in sequence and is sixteen amino acids longer than that encoded by the N-terminal exon of GKα. The crystal structures of recombinant GKαβ and GKββ from Namalycastis sp. were determined at 2.6 Å and 2.4 Å resolution, respectively. In addition, the structure of the GKββ was determined at 2.3 Å resolution in complex with a transition state analog, Mg2+-ADP-NO3--glycocyamine. Consistent with the sequence homology, the GK subunits adopt the same overall fold as that of other phosphagen 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 GKαβ are structurally defined and the longer N-terminus of the β subunit is anchored at the dimer interface. In GKββ 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 GKαβ amino acids involved in the interface formation were optimized once a heterodimer emerged as the physiological form of the enzyme. As a consequence, the homodimer interface (either solely α or solely β chains) has been corrupted. In the unbound state, GK exhibits an open conformation analogous to that observed with ligand-free CK or AK. Upon binding the transition state analog, both subunits of GK undergo the same closure motion that clasps the transition state analog, in contrast to the transition state analog complexes of CK, where the corresponding transition state analog 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 in both active sites of the GK heterodimer, 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 in the case of GK.

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confidence: 72%

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

et al. 2010

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“…tRNA was prepared according to [10]. Oxidation of tRNA was carried out as previously described [13]. tRNA(-1), tRNA(-2) and tRNA(-3) were prepared by subjecting tRNA to three reaction cycles of the Whitfield degradation according to Paulsen and Wintermeier [14] and purified using reverse phase chromatography [15].…”

Section: Methodsmentioning

confidence: 99%

Interaction of primer tRNA^Lys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

et al. 1995

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In the interaction between HIV-1 RT and tRNA Lys3 each subunit of the heterodimer interacts with tRNA showing a different affinity: Kd (p66) = 23 nM, Kd (pS1) = 140 nM. Preincubation of heterodimeric RT with tRNA, at concentrations similar to that of the Kd value for p51, leads to an increase of the catalytic activity on poly(A)-oligo(dT). These results were compared to those using different tRNA analogs: oxidized tRNA, tRNAs lacking one, two or three nucleotides from the 3'-end, or ribo-and deoxyribonucleotides mimicking the anticodon loop sequence. In all cases, tRNA analogs were weaker activators of HIV-1 liT than natural tRNA. A possible mechanism of RT p661p51 activation by tRNA and its analogs, mediated through the p5I subunit, is discussed.

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“…According to previous work, chemical modifications of this cysteine’s thiol generally either decrease the activity of CK or inactivate the enzyme completely. Additionally, several thiol-modifying reagents differentially modify one of the two active sites of the dimer, thus strongly suggesting that there is an intrinsic dimeric asymmetry that extends to the reactivity of the two thiols. − Measured enzymatic activity was decreased but not abolished upon mutation of Cys283 to either glycine, serine, alanine, asparagine, or aspartate; therefore, the Cys283 residue in rmCK (or the homologous Cys in other CK’s) is apparently optimal but not essential for catalysis or substrate binding. It was suggested that the cysteine residue might play a vital role in synergistic substrate binding, in which the binding of the first substrate (i.e., ATP) allows additional substrates (i.e., creatine) to bind with greater affinity to the CK active site before the reaction proceeds .…”

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confidence: 99%

Dynamic Asymmetry and the Role of the Conserved Active-Site Thiol in Rabbit Muscle Creatine Kinase

et al. 2014

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Symmetric and asymmetric crystal structures of the apo and transition state analogue forms, respectively, of the dimeric rabbit muscle creatine kinase have invoked an "induced fit" explanation for asymmetry between the two subunits and their active sites. However, previously reported thiol reactivity studies at the dual active-site cysteine 283 residues suggest a more latent asymmetry between the two subunits. The role of that highly conserved active-site cysteine has also not been clearly determined. In this work, the S-H vibrations of Cys283 were observed in the unmodified MM isoform enzyme via Raman scattering, and then one and both Cys283 residues in the same dimeric enzyme were modified to covalently attach a cyano group that reports on the active-site environment via its infrared CN stretching absorption band while maintaining the catalytic activity of the enzyme. Unmodified and Cys283-modified enzymes were investigated in the apo and transition state analogue forms of the enzyme. The narrow and invariant S-H vibrational bands report a homogeneous environment for the unmodified active-site cysteines, indicating that their thiols are hydrogen bonded to the same H-bond acceptor in the presence and absence of the substrate. The S-H peak persists at all physiologically relevant pH's, indicating that Cys283 is protonated at all pH's relevant to enzymatic activity. Molecular dynamics simulations identify the S-H hydrogen bond acceptor as a single, long-resident water molecule and suggest that the role of the conserved yet catalytically unnecessary thiol may be to dynamically rigidify that part of the active site through specific H-bonding to water. The asymmetric and broad CN stretching bands from the CN-modified Cys283 suggest an asymmetric structure in the apo form of the enzyme in which there is a dynamic exchange between spectral subpopulations associated with water-exposed and water-excluded probe environments. Molecular dynamics simulations indicate a homogeneous orientation of the SCN probe group in the active site and thus rule out a local conformational explanation at the residue level for the multipopulation CN stretching bands. The homogeneous simulated SCN orientation suggests strongly that a more global asymmetry between the two subunits is the cause of the CN probe's broad and asymmetric infrared line shape. Together, these spectral observations localized at the active-site cysteines indicate an intrinsic, dynamic asymmetry between the two subunits that exists already in the apo form of the dimeric creatine kinase enzyme, rather than being induced by the substrate. Biochemical and methodological consequences of these conclusions are considered.

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Functional non‐identity of creatine kinase subunits of rabbit skeletal muscle

Cited by 11 publications

References 7 publications

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

Interaction of primer tRNA^Lys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

Dynamic Asymmetry and the Role of the Conserved Active-Site Thiol in Rabbit Muscle Creatine Kinase

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Functional non‐identity of creatine kinase subunits of rabbit skeletal muscle

Cited by 11 publications

References 7 publications

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member,

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member,

Interaction of primer tRNALys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation

Dynamic Asymmetry and the Role of the Conserved Active-Site Thiol in Rabbit Muscle Creatine Kinase

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Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

Structural Basis for the Mechanism and Substrate Specificity of Glycocyamine Kinase, a Phosphagen Kinase Family Member^,

Interaction of primer tRNA^Lys3 with the p51 subunit of human immunodeficiency virus type 1 reverse transcriptase: a possible role in enzyme activation