Structural determinants of enzymatic processivity.
A processive enzyme binds a polymeric substrate and catalyzes a series of similar chemical reactions along that polymer before releasing the fully modified polymer to solvent. Bovine pancreatic ribonuclease A (RNase A) is a nonprocessive endoribonuclease that binds the bases of adjacent RNA residues in three enzymic subsites: B1, B2, and B3. The B1 subsite binds only to residues having a pyrimidine base, while the B2 subsite prefers adenine and the B3 subsite prefers a purine base. RNase A mutants were created in which all natural amino acids were substituted for Thr45 or Phe120, two residues of the B1 subsite. These pools of mutant enzymes were screened for mutants that catalyze the cleavage of RNA after purine residues. The Ala45 and Gly45 enzymes cleave poly(A), poly(C), and poly(U) efficiently and with 10(3)-10(5)-fold increases in purine/pyrimidine specificity. Thus, substrate binding can be uncoupled from substrate turnover in catalysis by RNase A. In addition, both mutant enzymes cleave poly(A) processively. Our results provide a new paradigm: a processive enzyme has subsites, each specific for a repeating motif within a polymeric substrate. Further, we propose that processive enzymes bind more tightly to motifs that do repeat than to those that do not.
delCardayré
S B
SB
Department of Biochemistry, University of Wisconsin-Madison 53706-1569.
Raines
R T
RT
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D011061:Poly A / Q000378:metabolism; D011066:Poly C / Q000378:metabolism; D011072:Poly U / Q000378:metabolism; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D011061:Poly A; D011072:Poly U; D011066:Poly C; D012259:Ribonuclease, Pancreatic