The coordination number of the catalytic zinc ion in alcohol dehydrogenase has been studied by integrated ab initio quantum-chemical and molecular mechanics geometry optimisations involving the whole enzyme. A four-coordinate active-site zinc ion is 100-200 kJ/mol more stable than a five-coordinate one, depending on the ligands. The only stable binding site for a fifth ligand at the zinc ion is opposite to the normal substrate site, in a small cavity buried behind the zinc ion. The zinc coordination sphere has to be strongly distorted to accommodate a ligand in this site, and the ligand makes awkward contacts with surrounding atoms. Thus, the results do not support proposals attributing an important role to five-coordinate zinc complexes in the catalytic mechanism of alcohol dehydrogenase. The present approach makes it possible also to quantify the strain induced by the enzyme onto the zinc ion and its ligands; it amounts to 42-87 kJ/mol for four-coordinate active-site zinc ion complexes and 131-172 kJ/mol for five-coordinate ones. The four-coordinate structure with a water molecule bound to the zinc ion is about 20 kJ/mol less strained than the corresponding structure with a hydroxide ion, indicating that the enzyme does not speed up the reaction by forcing the zinc coordination sphere into a structure similar to the reaction intermediates.
