Forcing enzyme activity

New research has shown that physically stretching an enzyme can trigger its activity - even when the active site is not hidden in a ’cryptic’ position. The work suggests that mechanical force may play a more important role in biological molecular systems than previously realised. 

It has been known for some time that enzymes with obscured (cryptic) binding sites can be activated by applying a force that exposes the active site. Now, however, Kerstin Blank from Radboud University in The Netherlands and her colleagues have demonstrated that a ’conventional’ enzyme can also be influenced by force. 

The researchers covalently anchored a yeast lipase enzyme to a glass slide. Above the enzyme they suspended a bead coated with an antibody to the enzyme and attached the bead to the tip of an atomic force microscope (AFM). When the bead is lowered the antibodies grab the enzyme. The AFM can then be raised, stretching the enzyme, until the antibody bond snaps, allowing the enzyme to relax and return to its original position. The enzyme can act on a substrate that releases a fluorescent product, whose presence can be measured by a technique called total internal reflection fluorescence (TIRF) microscopy - allowing the activity of the enzyme to be monitored during the whole process. 

The research team observed over a period of time a small amount of random residual activity by the enzyme before it was stretched. When the enzyme was stretched until the antibody bond broke, there was a distinct burst of activity around 1.7 seconds later. 

’The statistical distribution of activity is clustered around the stretching event,’ says Blank. ’It is clear that the force is causing activity to occur.’ 

The researchers speculate that the force which is applied to the enzyme pulls on a particular part of the molecule’s structure - a long alpha helix - which makes the enzyme’s active site more accessible to the substrate. 

’In general we think this work is important because so far studies on mechanical manipulation of enzymes have focused on exposing cryptic binding sites,’ Blank says. ’Our work shows that force might play a more general role in biological systems than we thought before. Also, using AFM together with fluorescence techniques could be more widely used in experiments to measure the effect of mechanical force on a range of other molecular systems.’ 

Nigel Scrutton, an eminent enzymologist based at the University of Manchester in the UK, says, ’The general approach will open up new studies of mechanical effects on enzyme activity and as such this is an important technological advance in the field.’ Scrutton adds, ’Of particular note is the clever use of antibody-antigen interactions as a ’molecular fuse’ to initiate relaxation following mechanical extension of the enzyme system. The method provides a new and exciting approach to study the conformational landscape of enzyme systems and how this relationship is linked to chemical catalysis.’ 

Simon Hadlington