Kinetics of the phosphotransferase reaction of the catalytic subunit of the tick salivary gland cAMP-dependent protein kinase

Journal Article · Thu May 01 00:00:00 EDT 1986 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:5023094

Mane, S D; Essenberg, R C; Sauer, J R

The catalytic subunit of the cAMP dependent protein kinase was purified 100-fold from tick salivary glands. The enzyme mechanism of the phosphotransferase reaction catalyzed by this subunit was investigated. Highly purified enzyme did not show ATP-ase activity in the absence of protein substrates. Initial velocities were measured using histone H-1 or a synthetic heptapeptide, Kemptide, as P/sub i/ acceptors and (..gamma..-/sup 32/P) ATP as a phosphodonor. Patterns were consistent with a sequential, but not a ping pong mechanism. At high concentration (>2Km), histone showed substrate inhibition which was noncompetitive versus ATP. Product inhibition by Mg.ADP was competitive versus ATP and noncompetitive with respect to H-1. Phosphohistone on the other hand was noncompetitive with respect to H-1, but gave parabolic competitive inhibition against ATP. Dead-end inhibition by AMP-PNP, an analogue of ATP, was competitive and noncompetitive against ATP and H-1, respectively. The inhibitory of cAMP dependent protein kinase was noncompetitive with ATP and competitive with histone. These studies strongly suggest that the tick salivary gland protein kinase has a sequential mechanism with primarily ordered addition of ATP followed by protein substrate and ordered release of phosphoprotein and ADP, but some random character.

• Research Organization: Oklahoma State Univ., Stillwater
• OSTI ID: 5023094
• Report Number(s): CONF-8606151-; TRN: 86-034803
• Journal Information: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PHOSPHOTRANSFERASES
BIOCHEMICAL REACTION KINETICS
PURIFICATION
ADP
ATP
HISTONES
PHOSPHORUS 32
TRACER TECHNIQUES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LIGHT NUCLEI
NUCLEI
NUCLEOPROTEINS
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
TRANSFERASES
550201* - Biochemistry- Tracer Techniques