Question

1. Compare the primary role of Mg²⁺ [ Mg(II)] in the mechanism of restriction endonucleases with that of Zn²⁺ in the carbonic anhydrases.
2. Explain the role of Ser 236 in abstracting a proton from the attacking water and giving up a proton to the Mg²⁺-ATP complex.

Answer 1

Carbonic anhydrases catalyze the reaction of water with carbon dioxide to generate carbonic acid. The catalysis can be extremely fast: molecules of some carbonic anhydrases hydrate carbon dioxide at rates as high as 1 million times per second. A tightly bound zinc ion is a crucial component of the active sites of these enzymes. Each zinc ion binds a water molecule and promotes its deprotonation to generate a hydroxide ion at neutral pH. This hydroxide attacks carbon dioxide to form bicarbonate ion, HCO₃^-. Because of the physiological roles of carbon dioxide and bicarbonate ions, speed is of the essence for this enzyme. To overcome limitations imposed by the rate of proton transfer from the zinc-bound water molecule, the most active carbonic anhydrases have evolved a proton shuttle to transfer protons to a buffer.

Within the enzyme-substrate complex, the DNA substrate is distorted in a manner that generates a magnesium ion binding site between the enzyme and DNA. The magnesium ion binds and activates a water molecule, which attacks the phosphodiester backbone.

Some enzymes discriminate between potential substrates by binding them with different affinities. Others may bind many potential substrates but promote chemical reactions efficiently only on specific molecules. Restriction endonucleases such as EcoRV endonuclease employ the latter mechanism to achieve levels of discrimination as high as million-fold. Structural studies reveal that these enzymes may bind nonspecific DNA molecules, but such molecules are not distorted in a manner that allows magnesium ion binding and, hence, catalysis. Restriction enzymes are prevented from acting on the DNA of a host cell by the methylation of key sites within their recognition sequences. The added methyl groups block specific interactions between the enzymes and the DNA such that the distortion necessary for cleavage does not take place.

Answer: The active site of myosin contains a group of highly conserved amino acid residues whose roles in nucleotide hydrolysis and energy transduction might appear to be obvious from the initial structural and kinetic analyses but become less clear on deeper investigation. One such residue is Ser236 (Dictyostelium discoideum myosin II numbering) which was proposed to be involved in a hydrogen transfer network during γ-phosphate hydrolysis of ATP, which would imply a critical function in ATP hydrolysis and motility. The S236A mutant protein shows a comparatively small decrease in hydrolytic activity and motility, and thus this residue does not appear to be essential. To understand better the contribution of Ser236 to the function of myosin, structural and kinetic studies have been performed on the S236A mutant protein. The structures of the D. discoideum motor domain (S1dC) S236A mutant protein in complex with magnesium pyrophosphate, MgAMPPNP, and MgADP·vanadate have been determined. wild-type S1dC, the S236A·MgAMPPNP complex crystallized in the closed state. Furthermore, transient-state kinetics showed a 4-fold reduction of the nucleotide release step, suggesting that the mutation stabilizes a closed active site. The structures show that a water molecule approximately adopts the location of the missing hydroxyl of Ser236 in the magnesium pyrophosphate and MgAMPPNP structures. This study suggests that the S236A mutant myosin proceeds via a different structural mechanism than wild-type myosin, where the alternate mechanism is able to maintain near normal transient-state kinetic values.