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| Type |
Oral Presentation |
| Area |
Oral Presentation of Young Analytical Chemists Ⅰ |
| Room No. |
303호 |
| Time |
THU 09:36-: |
| Code |
ANAL1.O-11 |
| Subject |
Substrate Binding Studies of Human Cytosolic Adenylate Kinase1 by NMR |
| Authors |
김길훈, 원호식*
한양대학교 응용화학과, Korea
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| Abstract |
Arginine residues are known to play important roles in substrate binding through the electrostatic interaction of positive charge on the Arg side chain with the negative charge of phosphoryl group in many of nucleotide binding proteins. Human cytosolic adenylate kinase1(hAK1) has some Arg residues necessary for binding its substrates including MgATP and AMP. To investigate the interactions between Arg side chains and the substrate in aqueous solution, we observed the signals of Arg ε-NHs of 15N-labeled AK using 1H, 15N two dimensional NMR. The experiments with SQC pulse sequence detected 13 signals corresponding to all Arg residues on hAK1(human cytosolic AK1). The 6 residue signals (Arg[44], [97], [128], [132], [138], and [149]) playing important roles in substrate binding associated with LID and AMP binding domain out of all corresponding 13 Arg residues were selectively assigned through Arg to Ala substitution. In kinetic studies, the resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites. Two substrates, MgATP and AMP were titrated into wild type hAK1 and mutants to observe the effects on these assigned signals. The addition of AMP affected 4 Arg signals, whose chemical shift took place a down field in the direction on proton dimension whereas the addition of MgATP did few effects. We were able to find that the Arg[44] specifically interact with the phosphoryl group of AMP. The additions of both the substrates augmented significantly the intensity of Arg[149] signal, which had very low intensity on the spectrum of free wild type AK1. Three ε-NH signals derived from Arg[123], [132], and [138] were broadened and disappeared upon the Mg-ATP addition. These arginine residues locate on α-helix and the following loop along the moiety of triphosphate of MgATP. |
| E-mail |
kghpotter@naver.com |
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119th General Meeting of the KCS