Chemistry and mechanism of vanadate-promoted photooxidative cleavage of myosin.
Irradiation of the stable myosin subfragment 1(S1).MgADP.orthovanadate (Vi) complex results in oxidation of an active site serine (Ser-180) to a serine aldehyde [Cremo, C. R., Grammer, J. C., & Yount, R. G. (1989) J. Biol. Chem. 264, 6608-6611]. This photomodified S1 will reform a new MgADP.Vi complex and upon a second irradiation, the S1 heavy chain is cleaved into 21 kDa NH2-terminal and 74 kDa COOH-terminal fragments. When S1, in which the side chain of Ser-180 was tritiated, was photocleaved tritium was released from the protein suggesting that cleavage was occurring at Ser-180. The 21 kDa NH2-terminal fragment was resistant to carboxypeptidase digestion, and the 74 kDa COOH-terminal fragment yielded no sequence by Edman degradation, indicating that parts of Ser-180 went to each fragment. To identify these parts, the two cleavage fragments were isolated and chemically (21 kDa) or enzymatically (74 kDa) cleaved, and the resulting peptides were separated by reversed phase HPLC. The peptides immediately down- and up-stream from Ser-180 were isolated and the blocking groups were identified by mass spectrometry. The 21 kDa fragment peptide was blocked with a carboxamide on Glu-179 (confirmed by HPLC and capillary electrophoresis in comparison with peptide standards), while the NH2 group of Gly-181 of the 74 kDa fragment was blocked with an oxalyl group (verified by enzymatic analysis for oxalate). The side chain of Ser-180 was released as formate. O2 is required for photocleavage. Cleavage experiments in the presence of 18O2 showed one atom of 18O labeled the oxalyl group. A mechanism in which O2 adds to a free radical on the alpha-carbon of Ser-180 with a subsequent Criegee type rearrangement is proposed to explain both the kinetics and products of the photocleavage.
Grammer
J C
JC
Department of Biochemistry and Biophysics, Washington State University, Pullman 99164-4660, USA.
Loo
J A
JA
Edmonds
C G
CG
Cremo
C R
CR
Yount
R G
RG
D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D007700:Kinetics; D013058:Mass Spectrometry; D018482:Muscle, Skeletal / Q000737:chemistry; D009218:Myosins / Q000378:metabolism*; D010084:Oxidation-Reduction; D010777:Photochemistry; D012694:Serine; D014638:Vanadates / Q000494:pharmacology*
D014638:Vanadates; D012694:Serine; D009218:Myosins