Mutational Analysis of Substrate Recognition by Protein Phosphatase 1
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- 作者：Lifang Zhang and Ernest Y. C. Lee
- 刊名：Biochemistry
- 出版年：1997
- 出版时间：July 8, 1997
- 年：1997
- 卷：36
- 期：27
- 页码：8209 - 8214
- 全文大小：138K
- 年卷期：v.36,no.27(July 8, 1997)
- ISSN：1520-4995
文摘
The role of residues that are involved in substrate recognition byrabbit muscle proteinphosphatase 1
(PP1) was investigated by site-directed mutagenesisand kinetic analyses usingphosphorylase a, RII peptide, Kemptide, andp-nitrophenyl phosphate as substrates. The atomicstructureof PP1 has shown the active site to be at the confluence of threeshallow grooves, a C-terminal groove,an acidic groove, and a hydrophobic groove. Mutations of residuesD208, D210, D212, E218, D220,E252, D253, E256, E275, and D277 in the acidic groove, of R221, W206,and Y134, which have beensuggested to be involved in substrate binding, and of residues C127,I130, and D197 in the hydrophobicgroove were examined. Our results show that mutations in theacidic groove lead to modest changes insubstrate binding, consistent with a role of the acidic residues informing a negatively charged surfacewell for binding of peptides with basic N-termini. Severe effectson Vmax were observed for mutantsofR221, D208, and W206. These results are consistent with theproposal that the R221 plays an importantrole as a phosphate oxygen ligand that positions the substrate forcatalysis. The kinetic behavior of mutantsat W206 and D208 can be explained by the observation that, togetherwith R221, these residues form themicroenvironment which dictates the orientation of the imidazole ringof H248, one of the metal bindingligands, as well as contributing to the orientation of R221itself.
(PP1) was investigated by site-directed mutagenesisand kinetic analyses usingphosphorylase a, RII peptide, Kemptide, andp-nitrophenyl phosphate as substrates. The atomicstructureof PP1 has shown the active site to be at the confluence of threeshallow grooves, a C-terminal groove,an acidic groove, and a hydrophobic groove. Mutations of residuesD208, D210, D212, E218, D220,E252, D253, E256, E275, and D277 in the acidic groove, of R221, W206,and Y134, which have beensuggested to be involved in substrate binding, and of residues C127,I130, and D197 in the hydrophobicgroove were examined. Our results show that mutations in theacidic groove lead to modest changes insubstrate binding, consistent with a role of the acidic residues informing a negatively charged surfacewell for binding of peptides with basic N-termini. Severe effectson Vmax were observed for mutantsofR221, D208, and W206. These results are consistent with theproposal that the R221 plays an importantrole as a phosphate oxygen ligand that positions the substrate forcatalysis. The kinetic behavior of mutantsat W206 and D208 can be explained by the observation that, togetherwith R221, these residues form themicroenvironment which dictates the orientation of the imidazole ringof H248, one of the metal bindingligands, as well as contributing to the orientation of R221itself.