Question

Ala Ala N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide subtilisin p-nitroaniline (bright yellow) OH uestions . Following Figure 5.1: Enzymatic assay for subtilisin activity. purification of the subtilisin S41 enzyme, the investigators carried out analytical procedures to determine the protein's molecular weight and pl. a. What technique would they have used to determine the protein's molecular weight? What technique would they have used to determine the protein's pl? psychrophilic subtilisin S41 enzyme was determined to be 5.3. This value is much lower than the pl of mesophilic subtilisins, which have pl values ranging from 9-11. Which amino acid residues does the subtilisin S41 have in greater amounts than the mesophilic subtilisins? The investigators then carried out an experiment in which they incubated subtilisin S41 and subtilisin Carlsberg (a mesophilic subtilisin) at 50EC and measured enzymatic activity as a function of time. The results are shown in Table 5.1. Interpret these results and explain their significance.

Answer 1

By using isoelctrofocusing protein’s pI can be determined.

Isoelectric point is the sum of total of pKa values of the side chain of acidic and basic amino acids amino acids. In this some amino acids such as Histidine is also participate.

As given question that psychrophilic subtilisins enzyme having pI is 5.3. In this pI is indicating the range below to neutral, pH 7, that indicate most of the acidic amino acids will participate means this protein contain more acidic amino acids. But pI is sum of the pKa values of side chain and hsitindine is 6.0 hence this amino acid will also participate.

pKa values of side chain of amino aids.

Asp=3.6

Glu= 4.2

Lysine =10

Arginine =12

Histindien= 6