Structure and activity of the tyrosy1-tRNA synthetase: the hydrogen bond in catalysis and specificity

Abstract

The role ofhydrogen bonding in specificity, binding and catalysis by the tyrosyl-tRNA synthetase from Bacillus stearothermophilus has been investigated by systematic mutation of residues which form hydrogen bonds with substrates during the reaction between ATP and tyrosine to form tyrosyl adenylate. Data on hydrogen bonding as a determinant of biological specificity are summarized thus: deletion of an hydrogen-bond donor or acceptor between the enzyme and substrate to leave an unpaired but uncharged acceptor or donor weakens binding by only 2-7 kJ mol ^-1 ; but deletion to leave an unpaired but charged acceptor or donor weakens binding by some 17 kJ mol ^-1 or so. Hydrogen bonding is found to have a profound role in catalysis by mediating the differential binding of substrates, transition states and products. The formation of tyrosyl adenylate is not catalysed by classical mechanisms of acid-base or nucleophilic catalysis but the enhancement of rate is solely a result of a combination ofhydrogen bonding and electrostatic interactions which stabilize the transition state of the substrates relative to their ground states. The binding energy of ATP increases by more than 29 kJ mol ^-1 as it passes through the transition state, enhancing the rate by more than a factor of 10 ⁵ . The residues involved in differential binding are spread over the molecule, away from the seat of reaction. The catalysis is delocalized over the whole binding site and not restricted to one or two specific residues. Some regions of the binding site are complementary in structure to the intermediate, tyrosyl adenylate. The apparent binding energies of certain side chains increase as the reaction proceeds, being weakest in the enzyme—substrate complex, stronger in the enzyme-transition-state complex and strongest in the enzyme-intermediate complex. This converts the unfavourable equilibrium constant for the formation of tyrosyl adenylate in solution to a favourable value for the enzyme-bound reagents and helps sequester the reactive tyrosyl adenylate.