| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 2001: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
It is well known that native peptidases and their mutants show bell-shaped pH dependent activity. From the simulation of log(k_<cat>/K_m)-pH plots, several reaction mechanisms were proposed, and the meaning of the pK_a values that define the plots were discussed ; however, the values were not corroborative because of the lack of effective experiments. It is difficult to obtain the pKa of Zn-OH_2 in the active site, where Zn-OH_2, substrate, and other proton-dissociative groups such as Im and -C00^-are forming complex hydrogen bonding knot. In the present study, we simplified the subject from enzyme to Zn^<2+> -containing short peptides in order to derive the pK_a of Zn-OH_2. For this purpose, we focused our attention on the motif sequence of Metzincin-clan peptidases, which has an eleven-residue sequence HE-X_2-H-X_2-G-X_2-H in the catalytic zinc-binding site.
Kinetic studies on AHEITHAVGMEHP/Zn^<2+> using equimolar amount of p-nitrophenylphosphate (p-NPP) as substrate in H_2O/DMSO = 1:
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1 indicated that the motif-peptide system obeys a step-wise mechanism represented by
E+S【double arrow】^^<k+1>__<k-1> E・S →^^<k+2> E+P1+P2 →^^<k+3> E・P1 (1)
This study afforded a well defined log(k_<+2>/K_m)-pH plot, from which pK_a = 8.0 and 9.3 were derived as those indicating the pH window of the hydrolase activity of this system. In order to clarify the pK_a of the imidazole NH protons, pH-dependent chemical shifts of imidazole CH(δ) were studied by NMR, and then based on the pK_a values thus obtained, potentiometry experiments were performed on MTZ13/Zn^<2+>. Potentiometry afforded pK_a= 8.0 for (H_4MTZ13)Zn-(H_20)_2 【double half arrows】 (H_4MTZ13)Zn-(H_2O)(OH) and 9.5 for (H_4MTZ13)Zn(OH)_2 【double half arrows】 (H_3MTZ13)Zn-(HO)_2. Consequently, our study showed that zinc hydroxide model is also available to our motif peptide systems.
In order to increase the hydrophobicity of surrounding media under aqueous conditions, thereby to avoid precipitation and to suppress conformational fluctuation due to hydrogen-bond exchange, we applied micellar systems for MTZ13/Zn^<2+>. CD experiments using cetyltrimetylammonium chloride afforded well defined spectra suggestive of robust structures (helicity > 60 %). Preliminary fluorescence studies using motif peptides with Trp in the sequence showed that the motif peptides are buried in the hydrophobic area of micelles. Less
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