There are five known families of carbonic anhydrases (a, b, g, d and z) that catalyze the reaction, CO 2 + H 2 O - HCO 3 - + H + a-Carbonic...

1.    There are five known families of carbonic anhydrases (a, b, g, d and z) that catalyze the reaction,

CO2 + H2O <-> HCO3- + H+

a-Carbonic anhydrase is found in humans and all other animals. b-Carbonic anhydrase is found in higher plants and certain bacteria. Unlike the a form, the b form is an allosteric enzyme that is regulated by a bicarbonate ion (also a product of the reaction). Given this information, complete the following questions.

a.    Both a- and b-carbonic anhydrases follow a ping-pong mechanism in which the first substrate bound by the enzyme is CO2. In what order are the other molecules in the reaction (H2O, HCO3-, H+) bound or released by the enzyme?

b.    Most b-carbonic anhydrases are composed of a dimer-of-dimers structure, i.e. a tetramer made up of two dimers. In one specific type of tetrameric b-carbonic anhydrase (Rv3588), a large number of basic and acidic amino acid residues interact on the interface between the dimers.

                                              i.    Which type of protein structure is a dimer of dimers: primary, secondary, tertiary, or quaternary? 

                                            ii.    Which type of noncovalent interactions are responsible for the two dimers binding to form a tetramer? Explain your answer.

c.    Consider the following figure illustrating T- and R-states of b-carbonic anhydrase.

                                              i.    Which state (T or R) does the bicarbonate ion bind and stabilize as an allosteric effector? How does bicarbonate stabilize this state? (i.e., identify the type of noncovalent interactions and amino acid residues involved in this stabilization)

                                            ii.    Which line, B or C, in the graph below best represents the effect of bicarbonate on b-carbonic anhydrase kinetics? Explain your answer.

                                           iii.    Consider when the Trp39 residue is mutated to a Phe (i.e., W39F). What effect would this mutation have on the T-R equilibrium? (i.e., would it shift it toward the T or R state?) Explain your answer.

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