Effect of diethyl pyrocarbonate modification on the calcium binding mechanism of the sarcoplasmic reticulum ATPase.
Diethyl pyrocarbonate was used to modify histidyl residues on the sarcoplasmic reticulum ATPase. Difference spectra of the N-carbethoxyhistidyl derivative indicated that most all the histidyl residues on the enzyme had been modified. These residues could be divided into two populations on the basis of their reaction rate with the reagent. It could then be shown that enzyme inhibition followed modification of the slower reacting population. Reversal with hydroxylamine verified that the loss of activity was due specifically to histidyl modification. Using [32P]ATP as a substrate it was further determined that the modified ATPase could form a phosphoenzyme intermediate, but that the hydrolysis of this intermediate was inhibited. Size exclusion chromatography was used to obtain equilibrium binding curves for high affinity Ca2+ sites on the enzyme. With the normal ATPase a cooperative binding curve for two Ca2+ with a Hill coefficient of 1.8 was observed. With the modified ATPase binding to two independent sites was observed; however, the dissociation constants remained the same as in the cooperative mechanism (K1 = 14 microM; K2 = 0.5 microM). That is, modification had eliminated cooperativity without changing the site specific binding affinities. E-P formation was then shown to follow binding to the higher affinity of the two sites. This would be the second site to bind Ca2+ in a sequential, cooperative mechanism. A model is suggested in which the binding of Ca2+ to an initial site allows for the binding of a second Ca2+ to an occluded site, this second site being responsible for enzyme activation. Modification apparently allows the binding properties of both sites to be observed independently.
Coan
C
C
Department of Physiology, School of Dentistry, University of the Pacific, San Francisco, California 94115.
DiCarlo
R
R
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors* / Q000302:isolation & purification / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002850:Chromatography, Gel; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D005561:Formates / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D005561:Formates; D006639:Histidine; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D004047:Diethyl Pyrocarbonate; D002118:Calcium