Abstract
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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.