Reactions of the sarcoplasmic reticulum calcium adenosinetriphosphatase with adenosine 5'-triphosphate and Ca/sup 2 +/ that are not satisfactorily described by an E/sub 1/-E/sub 2/ model

Journal Article · Tue Dec 01 04:00:00 EST 1987 · Biochemistry; (United States)

OSTI ID:5437991

Stahl, N; Jencks, W P

Phosphorylation of the sarcoplasmic reticulum calcium ATPase, E, is first order with k/sub b/ = 70 +/- 7 s/sup -1/ after free enzyme was mixed with saturating ATP and 50 ..mu..M Ca/sup 2 +/; this is one-third the rate constant of 220 s/sup -1/ for phosphorylation of enzyme preincubated with calcium of /sup c/E-Ca/sub 2/, after being mixed with ATP under the same conditions. Phosphorylation of E with ATP and Ca/sup 2 +/ in the presence of 0.25 mM ADP gives approx. 50% E approx. P-Ca/sub 2/ with k/sub obsd/ = 77 s/sup -1/, not the sum of the forward and reverse rate constants that is expected for approach to equilibrium if phosphorylation were rate limiting. These results show that (1) k/sub b/ represents a slow conformational change, rather than phosphoryl transfer, and (2) different pathways are followed for the phosphorylation of E and of /sub c/E-Ca/sub 2/. Chase experiments with unlabeled ATP or with ethylene glycol bis(..beta..-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) show that the rate constants for dissociation of (..gamma..-/sup 32/P)ATP and Ca/sup 2 +/ are comparable to k/sub b/. Approximately 20% phosphorylation occurs following an EGTA chase 4.5 ms after the addition of 300 ..mu..M ATP and 50 ..mu..M Ca/sup 2 +/ to enzyme. The calculated dissociation constant for Ca/sup 2 +/ from E-ATP-Ca/sub 2/ is approx. 2.2 x 10/sup -10/ M/sup 2/ (K/sub 0.5/ = 15 ..mu..M). It is concluded that steady-state turnover of the ATPase under most conditions occurs through the E-ATP-Ca/sub 2/ pathway, which has a relatively low affinity for Ca/sup 2 +/, not the pathway through /sup c/E-Ca/sub 2/ (or E/sub 1/-Ca/sub 2/). This results in 11-17% unphosphorylated enzyme in the steady state at saturating (ATP) and (Ca/sup 2 +/) because the k/sub b/step is partly rate limiting. The two pathways for phosphorylation can result in nonlinear Lineweaver-Burk plots for ATP and initial overshoots of phosphoenzyme levels.

• Research Organization: Brandeis Univ., Waltham, MA
• OSTI ID: 5437991
• Journal Information: Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 26:24; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
ALKALINE EARTH METAL COMPOUNDS
ANIMALS
ATP
ATP-ASE
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BIOLOGICAL PATHWAYS
CALCIUM COMPOUNDS
CATIONS
CELL CONSTITUENTS
CHARGED PARTICLES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DOSE-RESPONSE RELATIONSHIPS
ENZYMES
HYDROLASES
IONS
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MUSCLES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
RABBITS
RADIOISOTOPES
REACTION KINETICS
SARCOPLASMIC RETICULUM
VERTEBRATES