A couple questions from my chemistry class. Its an advanced college course and the professor made the questions that follow with all the intention of challenging anyone with chemical and biochemical background. Any help will be greatly appreciated.

1. Human-growth hormone binds to two different receptors, one of which also binds the hormone prolactin. Studies revealed that the growth-hormone-prolactin receptor interaction is weak in the presence of EDTA, but increases 8,000-fold upon the addition of 50 µM zinc. Mutagenesis studies indicated that changing His18, His21, or Glu174 in growth hormone to alanine reduced the strength of this interaction by two orders of magnitude. Even larger effects were observed following mutation of His188 in the prolactin receptor to alanine. Propose a model to account for these observations.

2. Suggest possible reasons why trivalent ions, such as Fe(III) or Mn(III), have not been widely utilized in hydrolytic metalloenzymes for substrate binding and activation.

3. Some proteases, such as chymotrypsin, do not contain metal ions in their active sites. Instead, there are several other functionalities that facilitate peptide bond cleavage. The reaction proceeds through nucleophilic attack of a serine hydroxyl on the peptide carbonyl group. The carbonyl group of the substrate is positioned near a cluster of peptide NH moieties. Compare the roles that these groups might play in catalysis with those of zinc in carboxypeptidase.

4. The cytochromes P450 are regarded as oxidizing enzymes; yet they consume one equivalent of NADH for each catalytic cycle. Why is NADH required? What atoms are formally reduced in the overall reaction?

5. The metal-binding selectivity of the copper protein azurin has been probed by using scanning calorimetry. In this method the temperature of a sample containing the protein is raised, and the amount of heat absorbed is monitored. At a certain temperature, corresponding to that at which the protein "melts", a large ampunt of heat is absorbed. For native, Cu(II) azurin, a total of 21 cal/g of heat is absorbed. For Ni(II) substituted azurin, only 10 cal/g are absorbed. Which metal is bound more tighty? Explain the difference in terms of ligand field stabilization.