I'm working with a beta-glycosidase from a hyperthermophilic archaeon. Eventhough it is highly thermostable, its kcat values are very low. So we tried to mutate some catalytic site and non catalytic site residues to increase its enzyme activity. Gln at 77th position (non-catalytic site residue) was replaced with Arg (Q77R). Q77R lost its activity by more than 90% when compared to wild-type. One possible reason might be the Gln involved in substrate binding. But, p-Nitrophenol B-galactopyranoside used for the hydrolytic assay is soluble and there is no substrate binding domain required. What could be the possible reason for the loss of activity by Q77R mutant.
Site-directed mutagenesis experiments can be difficult to interpret without structural information to show how the mutation affected the structure of the protein. You have change a neutral side chain to a charged one. This could have altered the structure or flexibility of the protein sufficiently to change kcat, Km or both even if the residue is not part of the active site.
By the way, when you measured the catalytic activity of this hyperthermophilic enzyme, did you do it at the temperature at which the organism normally grows? This is quite important, since the enzyme is adapted to work at a high temperature.
It is rare to find examples where researchers have improved enzyme activity(Kcat in particular) without some compensation in other aspects of the enzyme (Km, stability, oligomerization, etc). When you say Kcat is low, you are comparing it with something mesophilic enzyme, I assume.
1. Your enzyme might need some cofactor or condition that you are not able to provide in your reaction buffer.
2. The Kcat is good enough for the organism to thrive in its natural environment. It may be a slow growing organism and it may not need a high rate of catalysis.
3. You seem to assume that Km(roughly substrate affinity) has not changed in your Q77R mutant. A full enzymatic profile of the WT and the mutant/s has to be done to get a clear answer. When comparing enzymes make sure it is Kcat and not Vmax that you are comparing. I mean your protein has to be pure and your estimation of protein concentration accurate to make the case.
Your experiments will tell you the role of the glutamine residue (partly at least). Make Q77A and Q77E mutants and you should get an idea about it. As far as I know, protein chemists can predict quite well how enzyme activity can be compromised, but improving activity requires some trial/error followed by selection.
Site-directed mutagenesis experiments can be difficult to interpret without structural information to show how the mutation affected the structure of the protein. You have change a neutral side chain to a charged one. This could have altered the structure or flexibility of the protein sufficiently to change kcat, Km or both even if the residue is not part of the active site.
By the way, when you measured the catalytic activity of this hyperthermophilic enzyme, did you do it at the temperature at which the organism normally grows? This is quite important, since the enzyme is adapted to work at a high temperature.
@Adam Shapiro, I measure the catalytic activity at the optimum temperature of the enzyme.
As Adam Shapiro pointed out, we need to know something about the enzyme structure to interpret this. If the Gln is near the active site, it may still be participating in substrate binding. However, assuming it is far away, it could affect the structure or perhaps dimerization/multimerization, if a multimer quaternary structure is important to activity (thermophilic enzymes are often multimers due to the added energy of stabilization from monomer binding, but not always). Did you test the temperature and pH optimum of the mutant enzyme? The gel filtration profile, light scattering, and/or CD would be useful to evaluate the structural changes.
As Sujay said you have to do a complete enzyme profile before you can have any idea. I suggest doing kcat/Km, Activation energy and then activation thermodynamic parameters. WHAT-if tool has an option called mutate a residue. That can shed some light. Instead of SDM Directed Evolution is a better strategy to enhance the activity of thermostable enzymes while retaining their stability. It is certainly possible to avoid activity-stability trade-off.
Hello Boopathi, you have replaced a polar hydrophilic residue, , with a polar, hydrophilic residue, bearing a . This could modify the 3D structure of your enzyme to a certain extent, owing to electrostatic effects, therefore negatively affecting its activity. Moreover, arginine is bulkier than glutamine, and also this difference could be taken into the due consideration. Perhaps a change gln->ser or gln-> thr could help you tho understand the role of gln-77 in the catalytic activity you are studying.
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