1(1) Metallo-enzymes (permanent strong binding, cofactors)contain tightly bound ions, Fe++, Fe+++, Cu++, Zn++, Mn++, Co++(2) Metal activated enzymes (transient intermediate binding)loosely bind metal ions from solution Na+, K+, Mg++, Ca++(3) Transporters, pumps, ion channels (weak interaction)ions interact transiently with binding sites to achieve high flux rates across membranes H+, Na+, K+, Mg++, Ca++, Cl-Cox, Lehninger Principles in Biochemistry, chapter 8Metalloproteins2Zinc binding equilibrium of carbonic anhydraseDissociation constant KD= 10nMH20 + CO2↔ HCO3-+ H+HisHisHisZn++OHHCO2HCO3-bond weakendFigure 15-5a X-Ray structures of human carbonic anhydrase. (a) Its active site in complex with bicarbonate ion.Voet and Voet, Biochemistry, chapter 153Proposed mechanism of plant beta-Carbonic AnhydraseRowlett, R. S., C. Tu, et al. (2002). "Kinetic characterization of wild-type and proton transfer-impaired variants of [beta]-carbonic anhydrase from Arabidopsis thaliana*1." Archives of Biochemistry and Biophysics 404(2 SU -): 197-209.Cox, Lehninger Principles in Biochemistry, chapter 13Calmodulin, a calcium binding regulatory protein4Intracellular calcium oscillations visualized by fluorescence dye Fura II Cox, Lehninger Principles in Biochemistry, chapter 13, figure 18(a) thymocytes stimulated by extracellular ATP(b) single hepatocyte measured upon epinephrine (adrenaline) stimulationFigure 18-19Schematic diagram of the Ca2+–CaM-dependent activation of protein kinases.Figure 18-16 X-Ray structure of rat testis calmodulin.Voet and Voet, Biochemistry, chapter 185Branden & Tooze, Introduction to Protein Structure, 2nded, chapter 2, figure 13“Ca/CaM” Ca-binding motif (EF hand) shown for troponin CCox, Lehninger Principles in Biochemistry, chapter 13, figure 19X-ray structure of calcium-calmodulin (Ca/CaM) complexwith centrally bound target peptide6Comparison of CaM conformations and of CaM/effector contactsa: crystal structure without target peptide;b: canonical effector binding using hydrophobic clamp around inhibitory peptide; c: extended CaM binding around 3 helices with Ca ions bound to N-terminal domainonlyd: anthrax oedema factor (EF) CaM binding in extended and Ca-free N-terminal domain. Drum et al., 2002, Nature 415:396A: calmodulin, CaM; B: troponin C, TnCC: predicted binding of CaM with peptideD: NMR structure of CaM bound to skMLCK peptideE: crystal structure of CaM bound to smMLCK peptideF: crystal structure of mutant CaM unable to bind peptideMLCK=myosin light chain kinase; sk=skeletal muscle; sm=smooth muscleScience, 258 (2 October 1992)7Ca/CaMCaCaCaCaChannelPump10 M-710 M-3inoutCaNote: this organellar pump is regulated by phospholamban and not calmodulinRegulation of internal calcium concentration Secondary structure elements and domain organizationSecondary structure elements and domain organizationNature, June 2000, 405:648Figure 20-24b X-Ray structure of the Ca2+–ATPase from rabbit muscle sarcoplasmic reticulum. (b) A schematic diagram of the structure viewed similarly to Part a.Voet and Voet, Biochemistry, chapter 208Nature, June 2000, 405:648Figure 20-24a X-Ray structure of the Ca2+–ATPase from rabbit muscle sarcoplasmic reticulum. (a) A tube-and-arrow diagram. Note: this organellar pump is regulated by phospholamban and not calmodulin Voet and Voet, Biochemistry, chapter 20Ca-calmodulin binding curveNote: calmodulin system is never in saturation under physiological conditions making it a true sensor of calcium concentration changes.Note: calmodulin system is never in saturation under physiological conditions making it a true sensor of calcium concentration changes.1µMFractional saturationCa concentration5µM10µMPhysiological range of Ca signaling1µMFractional saturationCa concentration5µM10µMPhysiological range of Ca
View Full Document
Unlocking...