Fast infrared spectroscopy of enzyme catalysis

Enzymes are complex macromolecular structures that catalyse almost all the reactions that occur in biology. They are immensely efficient and are believed to have evolved close to 'perfection' in terms of the specific role that they play in metabolism. Fundamental studies of enzyme reaction mechanisms using relatively simple model enzymes have proved very valuable, giving many detailed insights into the way enzymes function in chemical terms. The advent of X-ray crystallographic structures changed the perception, in that one could now see the details of how the atoms involved in the catalytic act are organised in space and at least graphically, propose a plausible reaction mechanism. These mechanistic sketches contain no quantitative information and so have been tested by the all or nothing method of deleting or changing supposedly essential catalytic groups to measure what effect that has. In order to understand catalysis in more depth, one needs to be able to build up an energy diagram that relates the reaction progress with the energy of the system at any given point along the pathway. Some good progress was made many years ago by varying the substrate structure and deducing what effect that had on the reaction rate. There was and still is, a lack of incisive physical techniques that could measure the structural properties of dynamic events in catalysis. This application serves to address this issue by developing the application of fast (microsecond) infrared spectroscopy to studies of the dynamics of enzyme catalysis. This method measures the rate at which groups of atoms vibrate and is very sensitive to the weak forces, such as hydrogen bonding, that are crucial in enzyme catalysis. Thus, where it is possible to focus on catalytic events in the active site of an enzyme, it is possible to gain detailed information about the reaction intermediates (lowest energy states) along the pathway. These are important, as are the transition states (TS) energies gained from kinetic studies, in the evolutionary or lab mutagenic optimisation of catalysis. For optimal catalysis the intermediates should not be too stable and the TS energies as low as possible. This ensures that intermediates are not trapped and the energy barriers do not obstruct passage along the reaction pathway. We propose to develop the application of a new form of very fast IR spectroscopy to the detailed study of the mechanism and reaction pathway of the simple model pancreatic protein digesting enzyme chymotrypsin. This will allow us to construct such an energy diagram. A fast measuring method that will provide detailed structural information is needed to follow the reaction catalysed by enzymes, since these typically takes place in the millisecond time range. Chymotrypsin was the subject of intense study some 25 years ago but is not now of topical biological interest. It is however, a splendid object for study at a deeper level permitted by new technology, since it is robust, inexpensive and can hydrolyse a wide range of peptide and ester substrates. We propose to define the intermediates for this reaction in detail using substrates of increasing size and specificity to generate a structure activity correlation at a more detailed level that has been attempted before. The apparatus constructed will have wide-ranging application in future studies of enzyme catalysis. It will be applicable wherever a suitable inactive caged substrate (that can be made biological active by exposure to light) can be synthesised. This will, for example, include all reactions involving ATP & the other nucleotide phosphates. The enzyme chosen for instrument development and calibration and also for study in its own right is an archetype for several hundred enzymes of topical biological importance, since all employ the same catalytic mechanism. The outcome of this work will be applied to the study of bacterial resistance to penicillin family antibiotics.

Infrared spectroscopy is capable of providing potent information concerning protein structure and enzyme catalysis. Because the photons are of low energy they can be used to probe weak interactions such as hydrogen bonding in enzyme-substrate complexes and protein-ligand interactions. The aim is to be able to measure these factors i.e. the chemistry and the structural aspects, of a working enzyme reaction in real time. FTIR can be used to measure reactions that complete in a few hundred milliseconds but this is not fast enough for many reactions where the half-time for the reaction can be approximately 1 millisecond. To probe these reactions a faster method is needed. One such method that has been applied with success in studies of protein folding is laser IR spectroscopy. This is fine if one knows the vibration frequency to interrogate e.g. in protein folding 1653 cm-1 for a-helix. For chemical processes, where there is strong interaction with the enzyme, one does not know to what frequency the laser should be tuned. Because of this the analysis is inefficient in material and time. If a complete spectrum can be gathered on a fast time-scale then one is able to follow both the chemistry and the protein conformational changes simultaneously. Step-scan provides complete spectra with a time resolution on the nanosecond scale depending only on the response time of the detector. Step-scan is combined with continuous flow of a caged substrate, which is decaged by 10Hz laser pulses. Spectral accumulation takes ca. 5 min but if the flow cell is truly micro-volume (80 nl) then the material requirement is modest. Chymotrypsin will be used as the model enzyme to test the system and to determine the properties of the acylenzymes in the catalysis of the hydrolysis of highly specific substrates having a half-reaction time for a single turnover of ca. 5 milliseconds. We have developed a facile route to caged tyrosine which decages efficiently & is not a substrate until decaged.