Anionic substitutes for catalytic aspartic acids in phosphoribulokinase.
Mutagenic substitution of the invariant D42 and D169 residues in phosphoribulokinase (PRK) with amino acids that contain neutral side chains (e.g., alanine or asparagine) results in large decreases in catalytic efficiency (10(5)- and 10(4)-fold for replacement of D42 and D169, respectively). To further evaluate the importance of anionic side chains at residues 42 and 169, substitutions of glutamic acid (D42E, D169E) and cysteine (D42C and D169C in an otherwise cysteine-free protein) have been engineered. All purified mutant enzymes bind the fluorescent alternative substrate trinitrophenyl-ATP and the allosteric effector NADH similarly to wild-type PRK. For D42E and D42C, V(max) exhibits substantial decreases of 135- and 220-fold, respectively. Comparable substitutions for D169 result in smaller effects; D169E and D169C exhibit decreases in V(max) of 39- and 26-fold, respectively. Thus, regardless of the type of substitution, changes at D42 more profoundly affect catalytic rate than do comparable changes at D169. Precedent with enzymes in which cysteine replaces an acidic residue suggests that oxidation of the thiolate to a sulfinate can convert low-activity cysteine mutants into enzymes with improved activity. Periodate oxidation of cysteine-free PRK results in a slight decrease in activity. In contrast, comparable treatment of D42C and D169C proteins increases activity by 5- and 7-fold, respectively. Thus, for reasonably efficient catalysis, PRK requires anionic character in the side chains of residues 42 and 169. The enzyme can, however, tolerate substantial structural and chemical variability at these residues.
Runquist
Jennifer A
JA
Department of Biochemistry, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226, USA.
Miziorko
Henry M
HM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D019943:Amino Acid Substitution; D000838:Anions; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D000838:Anions; D005456:Fluorescent Dyes; D011994:Recombinant Proteins; D009243:NAD; D001224:Aspartic Acid; D000255:Adenosine Triphosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C022390:phosphoribulokinase; D003545:Cysteine