A Glutamate-Substituted Mutant Mimics the Phosphorylated and Active Form of Guanylyl Cyclase-A
| Autor: | Lincoln R. Potter, William G. McDowell, Neil Michael Otto, Deborah M. Dickey |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Mutant Glutamic Acid Michaelis–Menten kinetics 03 medical and health sciences Humans Amino Acid Sequence Phosphorylation Receptor Peptide sequence Pharmacology chemistry.chemical_classification Alanine Dose-Response Relationship Drug 030102 biochemistry & molecular biology Articles NPR1 Enzyme Activation HEK293 Cells 030104 developmental biology Enzyme Biochemistry chemistry Guanylate Cyclase Mutation Molecular Medicine |
| Zdroj: | Molecular Pharmacology. 92:67-74 |
| ISSN: | 1521-0111 0026-895X |
| Popis: | Multisite phosphorylation is required for activation of guanylyl cyclase (GC)-A, also known as NPR-A or NPR1, by cardiac natriuretic peptides (NPs). Seven chemically identified sites (Ser-487, Ser-497, Thr-500, Ser-502, Ser-506, Ser-510, and Thr-513) and one functionally identified putative site (Ser-473) were reported. Single alanine substitutions for Ser-497, Thr-500, Ser-502, Ser-506, and Ser-510 reduced maximal velocity (Vmax), whereas glutamate substitutions had no effect or increased Vmax. Ala but not Glu substitution for Ser-497 increased the Michaelis constant (Km) approximately 400%. A GC-A mutant containing Glu substitutions for all seven chemically identified sites (GC-A-7E) had a Km approximately 10-fold higher than phosphorylated wild-type (WT) GC-A, but one additional substitution for Ser-473 to make GC-A-8E resulted in the same Vmax, Km, and EC50 as the phosphorylated WT enzyme. Adding more glutamates to make GC-A-9E or GC-A-10E had little effect on activity, and sequential deletion of individual glutamates in GC-A-8E progressively increased the Km. Double Ala substitutions for Ser-497 and either Thr-500, Ser-510 or Thr-513 in WT-GC-A increased the Km 23- to 70-fold but the same mutations in GC-A-8E only increased the Km 8-fold, consistent with one site affecting the phosphorylation of other sites. Phosphate measurements confirmed that single-site Ala substitutions reduced receptor phosphate levels more than expected for the loss of a single site. We conclude that a concentrated region of negative charge, not steric properties, resulting from multiple interdependent phosphorylation sites is required for a GC-A conformation capable of transmitting the hormone binding signal to the catalytic domain. |
| Databáze: | OpenAIRE |
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