Steady state kinetics of spleen tyrosine kinase investigated by a real time fluorescence assay.
Spleen tyrosine kinase (Syk) is a cytoplasmic tyrosine kinase that plays an important signaling role in several types of immune cells. To improve our understanding of the enzymology and activation mechanism of Syk, we characterized the steady state kinetics of Syk substrate phosphorylation. A new real time fluorescence kinase assay was employed that utilizes a nonnatural amino acid in the peptide substrate which undergoes an enhancement in fluorescence following phosphorylation. Characterizing the steady state kinetics using a Syk kinase domain construct [Syk(360-635)] revealed that Syk employs a ternary complex kinetic mechanism involving little cooperativity between substrate binding sites and a Km(ATP) of 36 +/- 5 microM and a Km(peptide substrate) of 4.4 +/- 0.9 microM. The order of substrate binding was determined to be either random or ordered with ATP binding first, as determined in substrate analogue inhibitor studies. Utilizing the real time capabilities of the fluorescence assay, we established that Syk demonstrates no lag phase in product formation. Furthermore, a Syk mutant lacking tyrosine in the activation loop (Syk Y525F,Y526F) exhibited activity identical to that of wild-type Syk. These two findings indicate that autophosphorylation of the activation loop of Syk does not regulate Syk(360-635) activity. We also compared the activity of Syk(360-635) to that of full-length Syk and revealed that Syk(360-635) is 10-fold more active, suggesting that residues outside the catalytic domain of Syk suppress kinase activity. The findings presented here provide the first kinetic description of the Syk enzyme mechanism.
Department of Biochemical Pharmacology, Roche Palo Alto LLC, 3431 Hillview Avenue, Palo Alto, California 94304, USA.
Eva
E
Papp
Joyce K Y
JK
Tse
Hoangdung
H
Ho
Sandra
S
Wang
David
D
Shaw
Simon
S
Lee
Jim
J
Barnett
David C
DC
Swinney
J Michael
JM
Bradshaw
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D000072377:Syk Kinase; D013997:Time Factors
D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; D047428:Protein Kinase Inhibitors; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase