Transition state structure of arginine kinase: Implications for catalysis of bimolecular reactions

Zhou, Guowei; Somasundaram, Thayumanasamy; Blanc, Eric; Parthasarathy, G.; Ellington, W. Ross; Chapman, Michael S.

doi:10.1073/pnas.95.15.8449

Cited by 239 publications

(381 citation statements)

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“…HTK Prediction of the three-dimensional structure by Swiss Model indicated that the structure of Siphonosoma HTK is very similar to that of Limulus AK (data not shown), whose crystal structure has been determined [25].…”

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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Section: Structural Basis For Catalytic Properties Of Siphonosomamentioning

confidence: 99%

Hypotaurocyamine kinase evolved from a gene for arginine kinase

2005

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“…The three-dimensional structure of Sabellastarte AK2 was generated by SWISS-MODEL based on the TSAC structures of Limulus AK and Torpedo CK, both of which have a similar substrate-binding site [14,15]. Then we estimated the position of the substrate arginine in Sabellastarte AK2 by overlapping the position of L-arginine in the TSAC structure of Limulus AK (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2). 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].…”

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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“…6 Although the crystal structures of several vertebrate creatine kinases are currently available, only a single AK has been structurally characterized so far, that of Limulus polyphemus (Horseshoe crab) AK. 7 We have previously characterized the molecular and biochemical characterization of the T. cruzi AK (TcAK) 8,9 and we report here the crystal structure of the ligand-free (open form) of the enzyme at 1.9 Å resolution.…”

Section: Introductionmentioning

confidence: 99%