Micro Biology > QUESTIONS & ANSWERS > University of Illinois, Urbana ChampaignMCB 354MCB354SP16_PS01_KEY VERY RELIABLE (All)
Question 1 Activity of the bacterial enzyme acetoacetate decarboxylase sharply declines below pH 6, suggesting there is an ionizable catalytic residue with pKa ~ 6. Using mutagenesis, this critical... catalytic residue was determined to be Lys-115. a) What is the typical pKa of a lysine side chain and what ratio of deprotonated to protonated lysine would be expected at pH 7.5 (standard pH inside a bacterium)? The typical pKa for the side chain of lysine is 10.5. H-H equation: pH = pKa + log([A-]/[HA]) Inside a bacterium: 7.5 = 10.5 + log([lys-NH2]/[lys-NH3+]) And the ratio [lys-NH2]/[lys-NH3+] = 10(7.5–10.5) = 0.001 i.e. a very small amount of lysine is deprotonated at cytoplasmic pH. b) For catalytic Lys-115, what is the ratio of deprotonated to protonated side chain at pH 7.5? H-H equation: pH = pKa + log([A-]/[HA]) For catalytic lysine: 7.5 = 6 + log([lys-NH2]/[lys-NH3+]) And the ratio [lys-NH2]/[lys-NH3+] = 10(7.5–6) = 32 i.e. most of the catalytic lysine is deprotonated at cytoplasmic pH. c) The neighboring residue at position 116 is also lysine. Explain how this perturbs the pKa of the catalytic lysine residue. Two adjacent positive charges experience electrostatic repulsion. This destabilizes the ionization state in which both lysines are protonated, shifting the equilibrium for at least one lysine towards the neutral form (i.e. reduces the pKa and makes it a stronger acid). d) Lys-116 is mutated to glutamate. How is the pKa of catalytic Lys-115 expected to change and why? Negatively charged glutamate will have an attractive electrostatic interaction with protonated Lys-115, stabilizing the protonated species. The pKa for Lys-115 will therefore increase tremendously and it will become a stronger base [Show More]
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