alpha-chymotrypsin and 5--(4-fluorosulfonylbenzoyl)adenosine

Inactivation of rat liver S-adenosylhomocysteine hydrolase by the site-directed reagent 5'-p-fluorosulfonylbenzoyladenosine (FSBA) is associated with the formation of a disulfide bond between Cys-78 and Cys-112 (Takata, Y., and Fujioka, M. (1984) Biochemistry 23, 4357-4362; Gomi, T., Ogawa, H., and Fujioka, M. (1986) J. Biol. Chem. 261, 13422-13425). To characterize the inactivation mechanism more precisely, the properties of four hydrolase proteins mutated at Cys-78 or Cys-112 were compared to those of the wild-type enzyme. When Cys-78 was mutated to either a serine or an alanine, proteins with greatly reduced enzymatic activity were obtained, large effects on kinetic constants were observed, and enzymatic activity was not affected by incubation with FSBA. When Cys-112 was mutated to either a serine or an alanine, the activity was similar to the wild-type protein, only small changes in the kinetic constants were observed, and the enzyme was inactivated more rapidly upon incubation with FSBA. FSBA inactivation of the C112A mutant protein was accompanied by the formation of a disulfide between Cys-78 and Cys-52. The data indicate that FSBA initially reacts with Cys-78 and that Cys-78 has an important role in the structure of the enzyme.

Topics: Adenosine; Adenosylhomocysteinase; Animals; Base Sequence; Chymotrypsin; Cysteine; DNA Primers; Hydrolases; In Vitro Techniques; Kinetics; Liver; Molecular Sequence Data; Mutagenesis, Site-Directed; Rats; Recombinant Proteins; Structure-Activity Relationship

5'-(p-(Fluorosulphonyl)[14C]benzoyl)adenosine (FSBA) was synthesized and used as a probe to study the ATP-binding site of smooth-muscle myosin light-chain kinase (MLCK). FSBA modified both free MLCK and calmodulin/MLCK complex, resulting in inactivation of the kinase activity. Nearly complete protection of the calmodulin/MLCK complex against FSBA modification was obtained by addition of excess ATP whereas MLCK activity alone was lost in a dose-dependent manner even in the presence of excess ATP. These results suggest that FSBA modified ATP-binding sites and ATP-independent sites, and the latter sites are protected by calmodulin binding. The results also suggest that the ATP-binding site is accessible to the nucleotide substrate regardless of calmodulin binding. The FSBA-labelled MLCK was completely proteolysed by alpha-chymotrypsin, and the 14C-labelled peptides were isolated and sequenced. The sequence of the labelled peptide was Ala-Gly-X-Phe, where X is the labelled residue. The sequence was compared with the known MLCK sequence, and the labelled residue was identified as lysine-548, which is located downstream of the GXGXXG motif conserved among ATP-utilizing enzymes.

Topics: Adenosine; Adenosine Triphosphate; Affinity Labels; Amino Acid Sequence; Animals; Binding Sites; Chromatography, DEAE-Cellulose; Chromatography, High Pressure Liquid; Chymotrypsin; Consensus Sequence; Gizzard, Avian; Kinetics; Lysine; Molecular Sequence Data; Muscle, Smooth; Myosin-Light-Chain Kinase; Peptide Fragments; Protein Kinases; Ribonucleotides; Sequence Homology, Amino Acid; Turkeys

(1) FSBA, an inhibitor of platelet shape change and aggregation, inhibits and reverses the binding of fibrinogen to washed platelets induced by ADP in the presence of calcium ion; (2) FSBA does not inhibit either aggregation of or fibrinogen binding to platelets treated with chymotrypsin; (3) additional evidence is provided that FSBA does not inhibit aggregation by stimulating cyclic AMP accumulation; (4) FSBA does not inhibit PGE1-induced cyclic AMP accumulation and has little ability to inhibit the action of ADP on cyclic AMP or the binding of the ADP analog [beta 32P] 2-MeSADP; and (5) these results provide additional support for the view that the effects of ADP on aggregation and on cyclic AMP are mediated by distinct receptors, one of which, designated "A", is labeled by FSBA; the second receptor designated "C", has selective affinity for 2-MeSADP.

Topics: Adenosine; Adenosine Diphosphate; Blood Platelets; Chymotrypsin; Cyclic AMP; Fibrinogen; Humans; In Vitro Techniques; Platelet Aggregation; Receptors, Cell Surface; Receptors, Purinergic; Thionucleotides