Role of amino‐acid residue 95 in substrate specificity of phosphagen kinases

Tanaka, Kumiko; Suzuki, Tomohiko

doi:10.1016/j.febslet.2004.07.061

Cited by 38 publications

(36 citation statements)

References 33 publications

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“…Clearly, Y89Q has more specificity for D-arginine. These results are consistent with the idea that the 89th amino acid is the key residue for guanidino substrate recognition [20,21].…”

Section: Amino Acid 89supporting

confidence: 82%

“…The predicted structure indicated that the L-or D-arginine substrate is close to L64 in the N-terminal flexible loop (GS region) and N320 on the C-terminal flexible loop. Y89, which is proposed to be the key residue for guanidino substrate recognition [20,21], is also near the substrate arginines.…”

Section: Resultsmentioning

confidence: 99%

“…Amino acid residue-89 is proposed to be one of the key residues for recognition of guanidino substrates in phosphagen kinases [20,21]. Multiple alignments of amino acid sequences clearly indicated that the residue is strictly conserved as a specific amino acid in each phosphagen kinase: R in CK, Y in typical AK, I in GK, H in TK, and K in LK [24] (see Fig.…”

Section: Amino Acid 89mentioning

confidence: 99%

“…While this residue does not appear to be directly involved in substrate binding in the TSAC structures of CK and AK, it is located close to the guanidine substrate-binding site [14,15]. Site-directed mutagenesis studies of this residue using rabbit CK and Einenia LK have shown that it has a significant effect on enzymatic activity and guanidino substrate specificity [20,21]. A recent report investigating Danio CK and Nautilus AK also showed that the 89th residue plays a key role in organizing the hydrogen-bond network between the four dynamic domains [25] of AK and CK, thereby helping provide an appropriate active center for high catalytic turnover [26].…”

Section: Amino Acid 89mentioning

confidence: 99%

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Identification of the key amino acid residues in Sabellastarte arginine kinase for distinguishing chiral guanidino substrates (d- and l-arginine)

Uda

Matumoto

Suzuki

2010

Journal of Molecular Catalysis B: Enzymatic

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Arginine kinase (AK, EC 2.7.3.3) is a phosphagen kinase widely distributed in lower and higher invertebrates. Sabellastarte indica AKs (AK1 and AK2) are the only AKs that use D-arginine in addition to L-arginine as a substrate. Sabellastarte indica AKs are therefore clearly unrelated to typical AK. The purpose of this work was to identify the amino acid residues responsible for distinguishing the chirality of D-and L-arginine. The three-dimensional structure of Sabellastarte AK2 was predicted using the SWISS-MODEL automated modeling server. Two amino acids, L64 in the N-terminal flexible loop and N320 in the C-terminal flexible loop, were found to be closest to the substrate arginine. We introduced several mutations around the predicted binding site of the substrate arginine. In the L64I mutant, the affinity for L-arginine was greatly increased (9.5-fold that of the wild-type), whereas its affinity for D-arginine was increased 2.9-fold, indicating that the L64I mutant enzyme was more specific for L-arginine. On the other hand, the L64V mutant showed a 1.7-fold decrease in affinity for L-arginine, but unchanged affinity for D-arginine. Thus the L64V mutant enzyme was more specific for D-arginine. Since the replacement of amino acid residue L64 by I or V significantly affected the affinity for L-arginine or D-arginine, it can be concluded that amino acid 64 is a key residue for distinguishing D-and L-arginine. Seven mutants at position 320 (N320H, Q, D, E, R, K and A) had considerably reduced enzymatic activity (0.05 to 52% of the wild-type). The reduction in enzyme activity was more significant when D-arginine was the substrate rather than L-arginine, except for N320D, suggesting that the carbonyl oxygen of the Asn320 side chain forms a hydrogen bond with the α-amino nitrogen attached to the asymmetric carbon of the D-arginine substrate. In addition, we found that even a residue remote from the guanidino substrate binding site, such as G54 and Y89 in Sabellastarte 3 AK2, significantly affected guanidino substrate specificity.4

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“…Clearly, Y89Q has more specificity for D-arginine. These results are consistent with the idea that the 89th amino acid is the key residue for guanidino substrate recognition [20,21].…”

Section: Amino Acid 89supporting

confidence: 82%

Section: Resultsmentioning

confidence: 99%

Section: Amino Acid 89mentioning

confidence: 99%

Section: Amino Acid 89mentioning

confidence: 99%

See 2 more Smart Citations

Identification of the key amino acid residues in Sabellastarte arginine kinase for distinguishing chiral guanidino substrates (d- and l-arginine)

Uda

Matumoto

Suzuki

2010

Journal of Molecular Catalysis B: Enzymatic

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“…In addition, the amino acid sequence around the GS region is highly conserved in a given phosphagen kinase. Our previous studies, using Nautilus AK, Stichopus AK, Danio CK and Eisenia LK [18,[27][28][29], showed that the introduction of amino acid mutations in the GS region greatly reduced their enzymatic activity.…”

Section: Structural Basis For Catalytic Properties Of Siphonosomamentioning

confidence: 99%

Hypotaurocyamine kinase evolved from a gene for arginine kinase

2005

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Hypotaurocyamine kinase (HTK) is a member of the highly conserved family of phosphagen kinases that includes creatine kinase (CK) and arginine kinase (AK). HTK is found only in sipunculid worms, and it shows activities for both the substrates hypotaurocyamine and taurocyamine. Determining how HTK evolved in sipunculids is particularly insightful because all sipunculid-allied animals have AK and only some sipunculids have HTK. We determined the cDNA sequence of HTK from the sipunculid worm Siphonosoma cumanense for the first time, cloned it in pMAL plasmid and expressed it in E. coli as a fusion protein with maltose-binding protein. The cDNAderived amino acid sequence of Siphonosoma HTK showed high amino acid identity with molluscan AKs. Nevertheless, the recombinant enzyme of Siphonosoma HTK showed no activity for the substrate arginine, but showed activity for taurocyamine. Comparison of the amino acid sequences of HTK and AK indicated that the amino acid residues necessary for the binding of the substrate arginine in AK have been completely lost in Siphonosoma HTK sequence. The phylogenetic analysis indicated that the HTK amino acid sequence was placed just outside the molluscan AK cluster, which formed a sister group with the arthropod and nematode AKs. These results suggest that Siphonosoma HTK evolved from a gene for molluscan AK. Moreover, to confirm this assertion, we determined by PCR that the gene for Siphonosoma HTK has a 5-exon/4-intron structure, which is homologous with that of the molluscan AK genes. Further, the positions of splice junctions were conserved exactly between the two genes. Thus, we conclude that Siphonosoma HTK has evolved from a primordial gene for molluscan AK.

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Cold-adapted Features of Arginine Kinase from the Deep-sea Clam Calyptogena kaikoi

et al. 2011

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The heterodont clam Calyptogena kaikoi, which inhabits depths exceeding 3,500 m where low ambient temperatures prevail, has an unusual two-domain arginine kinase (AK) with molecular mass of 80 kDa, twice that of typical AKs. The purpose of this work is to investigate the nature of the adaptations of this AK for functioning at low temperatures. Recombinant C. kaikoi AK constructs were expressed, and their two-substrate kinetic constants (k(cat), K(a), and K(ia)) were determined at 10°C and 25°C, respectively. When measured at 25°C, the K(ia) values were tenfold larger than those for corresponding K(a) values, while at 10°C, the K(ia) values decreased remarkably, but the K (a) values were almost unchanged. The Calyptogena two-domain enzyme has threefold higher catalytic efficiency, calculated by k (cat)/(K(a)(ARG)·K(ia)(ATP) ), at 10°C, than that at 25°C, reflecting adaptation for function at reduced ambient temperatures. The activation energy (E(a)) and thermodynamic parameters were determined for Calyptogena two-domain enzyme and compared with those of two-domain enzymes from mesophilic Corbicula and Anthopleura. The value for E(a) of Calyptogena enzyme were about half of those for mesophilic enzymes, and a larger decrease in entropy was observed in Calyptogena AK reaction. Although large decrease in entropy increases the ΔG(o‡) value and consequently lowers the k(cat) value, this is compensated with its lower E(a) value thereby minimizing the reduction in its k(cat) value. These thermodynamic properties, together with the kinetic ones, are also present in the separated domain 2 of the Calyptogena two-domain enzyme.

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Role of amino‐acid residue 95 in substrate specificity of phosphagen kinases

Cited by 38 publications

References 33 publications

Identification of the key amino acid residues in Sabellastarte arginine kinase for distinguishing chiral guanidino substrates (d- and l-arginine)

Identification of the key amino acid residues in Sabellastarte arginine kinase for distinguishing chiral guanidino substrates (d- and l-arginine)

Hypotaurocyamine kinase evolved from a gene for arginine kinase

Cold-adapted Features of Arginine Kinase from the Deep-sea Clam Calyptogena kaikoi

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