Question

Contrast substrate binding and catalytic activity as mechanisms for attaining substrate specificity in restriction endonucleases.

Answer 1

Many restriction endonucleases show relatively low degree of promiscuous activity under standard reaction conditions in the presence of magnesium ions but exhibit significant star activity under altered conditions namely low ionic strength, elevated pH or when magnesium ion is substituted with maganese ion. Kpn I enzyme however exhibits significant promiscuous activity in the presence of magnesium ion. The dramatic effect of metal ion cofactor on the KpnI activity is intriguing and suggestes that altering the active site interactions with the metal ion could affect the enzyme's specificity.

Altering the metal ion binding properties of KpnI and possibly other restriction endonucleases can alter its substrate specificity and reinforce the notion that bound metal ion is correlated to the promiscuous activity of the enzyme.