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106 Chapter 5
Figure 5.32 Structure of ribonuclease A (PDB Wle 1AFL), showing the catalytic residues His-12,
His-119 and Lys-41 in red, and a bound inhibitor in black.
pyrimidine 3 0 -phosphate product. Covalent modiWcation studies using iodoacetate
and iodoacetamide implicated two histidine residues, His-12 and His-
119, as being involved in catalysis. When the X-ray crystal structure of the
enzyme was solved, these residues were found on opposite sides of the active
site, with the side chain of Lys-41 also occupying a prominent position (see
Figure 5.32). The currently accepted mechanism for ribonuclease A is shown in
Figure 5.33. The mechanism involves participation of the 2 0 -hydroxyl of the
pyrimidine residue, which is deprotonated by His-112 and attacks the phosphodiester
via an associative mechanism to form a divalent transition state
stabilised by Lys-41. Breakdown of this transition state using His-119 as an acid
leads to a cyclic phosphodiester intermediate which, due to internal strain, is
much more reactive than the substrate. Acid–base catalysis by the two histidine
groups completes the mechanism via a second divalent transition state.
5.6 Adenosine 5 0 -triphosphate
Enzymatic phosphoryl transfer reactions usually involve the transfer of phosphoryl
groups from a ‘high-energy’ phosphoric anhydride species to an acceptor
which can be an alcohol, a carboxylic acid or another phosphate. The most
common source of phosphoryl groups for such transfer reactions is the coenzyme
adenosine 5 0 -triphosphate or ATP. This is the nucleoside triphosphate
derivative of adenosine, which is one of the components of RNA. However, in
addition to its role in RNA it is used as a coenzyme by a wide range of enzymes.