6) Mutation Analysis of Catalytic Mechanism and Substrate Specificity of Chymotrypsin & 7) synthesis of ds cDNA from mRNA

goal of protein biochemistry research

characterize a protein thru structure and function/mechanism

how do we know that asp 102 / his 57/ ser 195 are important to activity of chymotrypsin

if you change any of them, rate of reaction drops significantly

why is changing ser 195 and his 57 to ala more intense?

ser - directly involved in the intermediates being formed

his - helps form the intermediates thru deprotonation

substrate specificity- trypsin

trypsin cleaves arg and lys and has asp at AA 189 instead of ser (this difference at 189 is where substrate specificity comes into play)

D189S mutant significance

need a lot less substrate for rxn bc you’re essentially converting trypsin (which has significantly larger Km) to chymotrypsin by changing Asp at 189 to Ser

how to make mutations through gene cloning

1) isolate mRNA

2) convert mRNA into cDNA

3) insert cDNA into vector (plasmid)

4) transform cells

5) screen cells for gene of interest

6) sequence the vector that contains the gene of interest

how are mutations introduced in the gene

through QuickChange PCR mutagenesis

converting ss cDNA into ds cDNA

mRNA w Poly(A) tail + Oligo (T) primer through reverse transcriptase dNTPs → mRNA + cDNA through alkali digestion of mRNA template and attaching oligo(dG) to 3’ end of cDNA → through oligo(dC) primer + DNA polymerase dNTPs → produces ds cDNA

afterwards, add appropriate linkers (restrction enzyme sites ) to ends of ds cDNA