A kinetic analysis of the processive enzyme Lactobacillus plantarum exoribonuclease.

Oligonucleotide chains consisting of adenosine residues and ranging from 1 to 70 residues in length have been tested as substrates or inhibitors with Lactobacillus plantarum exoribonuclease (EC3.1.4.20). The kinetic constants V, Km, and Ki are all chain-length dependent. Ki decreases with increasing chain length to a minimum for oligonucleotides seven residues in length and then begins to increase slightly. Kinetic plots indicate that the oligonucleotides are almost all competitive inhibitors of poly A hydrolysis. However, the oligonucleotide (Ap)3A greater than p probably leads to mixed inhibition. The enzyme is unable to retain its processivity when it hydrolyzes short oligonucleotides such as (Ap)2A and (Ap)3A. It is proposed that L. plantarum exoribonuclease possesses seven binding sites for the polynucleotide. When the enzyme is bound to a long-chain-length substrate the complex is stabilized by a binding energy of about 8 Kcal/mol. After cleavage of the terminal nucleotide the remaining binding energy is still sufficient to maintain an enzyme-substrate complex. The shortened nucleotide chain is moved relative to the enzyme to re-form the seven-bond association by a gradient of energy of about 1.7 Kcal/mol for the change from six to seven bonds.