期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 287, 期 39, 页码 32717-32727出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M112.373472
关键词
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资金
- National Institutes of Health [RO1 HL69830]
- Ente Cassa di Risparmio di Firenze [2009.0749]
- Italian Ministry of Education, University and Research [20083YM37E]
Ca2+ (sarco-endoplasmic reticulum Ca2+ ATPase (SERCA)) and Cu+ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca2+, demonstrated by the addition of ATP and Ca2+ to SERCA deprived of Ca2+ (E2) as compared with ATP to Ca2+-activated enzyme (E1 center dot 2Ca(2+)). Activation by Ca2+ is slower at low pH (2H(+)center dot E2 to E1 center dot 2Ca(2+)) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca2+ translocation. A H+-gated pathway, demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca2+ release by H+/Ca2+ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu+/H+ exchange. As opposed to SERCA after Ca2+ chelation, ATP7A/B does not undergo reverse phosphorylation with Pi after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.
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