A designed peptide ligase for total synthesis of ribonuclease A with unnatural catalytic residues.
An engineered variant of subtilisin BPN', termed subtiligase, which efficiently ligates esterified peptides in aqueous solution, was used for the complete synthesis of ribonuclease (RNase) A that contains unnatural catalytic residues. Fully active RNase A (124 residues long) was produced in milligram quantities by stepwise ligation of six esterified peptide fragments (each 12 to 30 residues long) at yields averaging 70 percent per ligation. Variants of RNase A were produced in which the catalytic histidines at positions 12 and 119 were substituted with the unnatural amino acid 4-fluorohistidine, which has a pKa of 3.5 compared to 6.8 for histidine. Large changes in the profile of the pH as it affects rate occurred for the single and double mutants with surprisingly little change in the kcat for either the RNA cleavage or hydrolysis steps. The data indicate that these imidazoles function as general acids and bases, but that the proton transfer steps are not rate-limiting when the imidazoles are present in their correct protonation states. These studies indicate the potential of subtiligase for the blockwise synthesis of large proteins.
Jackson
D Y
DY
Department of Protein Engineering, Genentech, Inc., South San Francisco, CA 94080.
Burnier
J
J
Quan
C
C
Stanley
M
M
Tom
J
J
Wells
J A
JA
D000595:Amino Acid Sequence; D001665:Binding Sites; D004951:Esterification; D006639:Histidine / Q000031:analogs & derivatives / Q000032:analysis; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D009154:Mutation; D009712:Nucleotides, Cyclic / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D012259:Ribonuclease, Pancreatic / Q000138:chemical synthesis* / Q000737:chemistry / Q000302:isolation & purification; D013381:Subtilisins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014542:Uridine Monophosphate / Q000378:metabolism
C089806:4-fluorohistidine; D009712:Nucleotides, Cyclic; C045338:cyclic 3',5'-uridine monophosphate; D006639:Histidine; D014542:Uridine Monophosphate; D012259:Ribonuclease, Pancreatic; D013381:Subtilisins