Trypsin‐Catalyzed Synthesis of a Semisynthetic Aspartate Aminotransferase a

Existing experimental evidence indicates that the amino-terminal peptides of aspartate aminotransferase, which act as bridges between the subunits of this dimeric protein, are an essential element in the overall structural stability of the enzyme and participate in the conformational transitions that play an integral role in its catalytic Limited tryptic proteolysis of cytosolic aspartate aminotransferase results in cleavage of these peptides after Lys,,.' In the present studies, trypsin catalysis of peptide bond synthesis has been exploited to introduce selective modifications into the aminoterminal region of this protein. Semisynthesis reactions were performed using as substrates: native cytosolic transaminase and a 19-residue PLP-labeled and tritiated synthetic peptide corresponding to the native amino-terminal peptide in amino acid sequence, except that an arginine residue was substituted for Lys,,. Studies aimed at optimizing the efficiency of the semisynthesis reaction revealed that maximal yields of semisynthetic product were obtained when transaminase, trypsin, and synthetic peptide, in a molar concentration ratio of 1 : 1 : 10, were incubated at 37°C in 0.1 M Tris buffer containing 40% glycerol (vol/vol), a t pH,,, 7.2 (FIG. l), and when the reactions were kinetically controlled, being terminated after 9 to 10 hours of incubation. The addition of transamination substrates, known to inhibit trypsin hydrolysis of the amino-terminal peptide segment from the native enzyme, decreased the efficiency of the reaction. Semisynthetic transaminase was purified from reaction mixtures using G150 Sephadex and chromatofocusing column chromatography, with typical yields being 2% to 3% of the enzyme originally added to the reaction. Amino acid analysis of peptides released during limited chymotrypsin proteolysis of the purified semisynthetic enzyme identified it as PLP-(Arg,,)-aspartate aminotransferase (FIG. 2 ) . The structurally altered protein exhibited catalytic activity, heat stability, and susceptibility to proteolysis similar to that of the native enzyme. These results indicate that selective modifications can be introduced into proteaseaccessible amino-terminal regions of globular proteins by simultaneous protease