Baird et al. tested the activity of their threonine mutant protein. In other var
ID: 214877 • Letter: B
Question
Baird et al. tested the activity of their threonine mutant protein. In other variations, they also tested mutant protein between c are shown below for the normal "wild type" protein and various mutant proteins with two substrates. (note that "$195T" means that the serine at position 195 was changed to threonine, etc.) s from which they removed a neighboring disulfide bridge ystines 42-58 to reduce steric clashes with thr195. Their real experimental data Table 1. Activity of wild-type trypsin and variants Substrate Z-GPR-Sbzl Z-GPR-pNA kcat (min-|) (-1 min-i) (uM) (uM-1 mini) (mini) (uM) Variant 715.5 165 1.2 x 10 46.5 258.06 43.4 Wild type S195T C42A/C58A C42A/C58V C42A/C58A/S195T 121.5 C42A/C58V/S19ST 0.023 5.0 7.0 2.85 32.8 10.8 0.008 0.15 0.65 0.166 7.4 656.4 44.6 29.5 217.7 22.2 0.351 0.009 89 76. 3.5 0.73 4.79 2.0 214 Please use biochemical terms accurately, precisely, and specifically in each answer below. How do the structural changes relate to the observed functional changes? a. How well does the S195T mutant bind or cleave substrate Z-GPR-Sbzl (left side of table)? b. How well does the C42A/C58A/S195T mutant bind or cleave substrate Z-GPR-Sbzl?Explanation / Answer
How do the structural changes relate to the observed funcional changes?
Answer: The changes in the amino acid content affects directly the protein structure as well as the protein function by altered the kinetics of the enzyme.
a. In order to evaluate how well the mutant protein binds or cleaves to the substrate, you must pay attention to the value for KM (Michaelis constant) for the S195T, because this constant shows the affinity of the enzime to an especific substrate (in this case Z-GPR-Sbzl), so a small value for KM means a greater grade of affinity. As the table shows, the KM for S195T it is smaller than the value for the wild type enzyme, which indicates that this mutat protein has more affinity for Z-GPR-Sbzl substrate than the wild type protein.
b. As mencioted before, the value for KM it is the one we use to evalute the affinity of the C42A/C58A/S195T protein to the substrate Z-GPR-Sbzl, in the table it is reported a KM= 1.4, which it is smaller than the reported for the wildtype protein and the S195T mutant protein, therefore it has more affinity i.e. it binds better to the substrate mencited before than the other two.
c. It does not has the best affinity for the substrate because the value for KM it is the greatest of all the proteins tested, so it reflects a low rate of binding of the protein C42A/C58A for the substrate. the reason for which it was tested the proteins without the mutant S195T in order to evalute the impact of this mutant on the enzyme kinetics.
d. For all the proteins tested there is an increse in the value for KM that indicates more affinity of all proteins for the second substrate (Z-GPR-pNA) although the catalytic efficiency (kcat/KM) it is less than for the first substrate (letf) for the majority of the proteins except the wild type and the C42A/C58V.
e. Removing the disulfide bridges can alterate the tridimensional structure of the protein, as well as their response to heat, pH and polarity.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.