Regulation of the SERCA calcium pump by phospholamban (PLB) is largely due to 
interactions between their respective transmembrane domains. In spite of 
numerous mutagenesis and kinetic studies, we still do not have a clear 
mechanistic picture of how PLB influences the calcium transport cycle of SERCA. 
Herein, we have created alanine mutants for each residue in the transmembrane 
domain of PLB, we have co-reconstituted these mutants with SERCA into 
proteoliposomes, and we have performed kinetic simulations of the 
calcium-dependent ATPase activity isotherms. The PLB transmembrane mutants had a 
variable effect on the calcium affinity, maximal activity, and cooperativity of 
SERCA, such that a range of values was observed. Kinetic simulations using a 
well-established reaction scheme for SERCA then allowed us to correlate the 
effects on SERCA activity with changes in the reaction scheme rate constants. 
Only three steps in the reaction scheme were affected by the presence of PLB, 
namely, binding of the first calcium ion, a subsequent conformational change in 
SERCA, and binding of the second calcium ion. The ability of wild-type and 
mutant forms of PLB to alter the apparent calcium affinity of SERCA correlated 
with a decreased rate of binding of the second calcium ion. In addition, the 
ability of wild-type and mutant forms of PLB to alter the maximal activity of 
SERCA correlated with a change in the forward rate constant for the slow 
conformational change in SERCA following binding of the first calcium ion.