Metal ions or some chemicals can be effect on enzyme activity. In determining Km and Vmax values, firstly metal ion concentration or which metal ion must be known. This situation depends on your studying enzyme. You can be find necessary knowledge in the literature. And then you should characterize your enzyme.

This is a very important question. We really need to be looking at inverse micelle systems for enzymes, which gives a more realistic picture of how enzymes work in vivo. Your enzyme sounds like the ones I investigated a long time ago,that hydrolyzes glycolipids. I suspected that at least one of them, the beta-glucosidase deficient in Gaucher's disease, actually works swimming inside a membrane, where the amphiphilic substrate is also located. I don't know if there has been any progress on that in the intervening 30 years.

There is no simple answer to this question. My first thoughts were that a series of kinetic experiments should be performed beginning with the enzyme alone and then with the addition of the various activators both individually and in combination. However, the actions of triton and the organic solvents suggest that they are causing disruption/removal of lipid or release of the enzyme protein from a membrane. The detergent/solvent treatment would improve the exposure of the enzyme to the aqueous media. If such is the case, perhaps it is best to begin the study with enzyme that has been treated with triton and then elucidate the action of NaCl on the kinetic parameters. The triton treated preparation would hardly represent the physiological state of the enzyme however, if it is known to be membrane bound in vivo. Without knowing more about the enzyme, it is difficult to provide further advice.

Peter

Your interest should be to get the maximum activity of the enzyme. You add anything that will take you in this direction. Once you have reached a plateau then analyze what are the roles of each of these additives in maximizing the enzyme activity. Do they work independently or in combination.

It depends on the question you want to answer.

Are you trying to repeat previous work? If so, make sure to use the conditions described previously so your KM and Vmax will match the previous results.

Are you characterizing this enzyme for the first time? If so, as stated above, you really should perform some dependence studies to see what the optimal conditions. To do so, keep the concentration of everything in your assay (substrate, enzyme concentration, salt) constant as you vary the triton concentration. Then do the same thing, but keeping triton constant (and everything else) constant as you vary the salt concentration. I also recommend that you do this with your enzyme concentration, once you've worked out the other parameters, as you will want to use an enzyme concentration that lies within the linear range of a [enzyme] vs activity plot.

Do you want to know how triton/salt/solvents affect the KM and Vmax of your enzyme? This is a lot of work, but once the conditions from the last experiment are worked out, you can go about determining the kinetic parameters of your enzyme at specific concentrations of triton/salt/solvents to see how this causes KM and Vmax to change.

The absolute value you calculate for an in vitro enzyme assay will probably never be the same as the in vivo activity. The conditions you use all depend on what question you want to answer. If you are comparing WT enzyme to clinically relevant mutations, go with conditions that give the maximum activity so the mutant activity is high enough to be measured relative to background. If you want to compare your enzyme with another closely related enzyme who's activity has previously been reported, keep the conditions identical.

First, please check the enzyme-concentration dependence of the initial velocity.

because you want to know properties of your enzyme such as Km and Vmax, I think that another activator such as metal Ions can affected on your enzyme activity and the end effect on velocity of your enzyme and your result will not appear to real. you should use only Enzyme+Buffer+substrate that you should use a gradient for substrate concentration for Km and that you can analysis Vmax by Sigmaplot 10.

The usual case is to use certain concentration i.e 10 ug of your enzyme dissolved in in your acetate buffer against different concentrations of substrate dissolved in same buffer and go on ..... Calculate sp activity then reaction velocity then plot 1/s against 1/v deduce km and vmax then calaculate kcat

Additives are based on the enzyme application condition. If you are looking for absolute Km and Vmax just go for the basic elements those are necessary to have active enzyme. Good luck

Thanks everyone for all the wonderful answers, I'm working on a recombinant tetrameric protein in the cytoplasm. So it is actually surprising that triton enhances activity. I dont know if I'll have the time to study all the additives, and I need to compare the kinetic parameters with homologous enzymes. So I'm planning to start with similar basic parameters as the other enzyme and study how the additives affect the kinetic constants later on..