Catalase

Catalase Catalase is a heme containing redox enzyme. It is found in high concentrations in a compartment in cells called the peroxisome.

Decomposition of H2O2 Catalase disproportionates: 2H2O2   to   2H2O + O2, H2O2 is a powerful oxidizing agent and is potentially damaging to cells. By preventing excessive H2O2 build up Catalase allows important cellular processes which produce H2O2 as a byproduct to take place safely.

The Peroxidative Reaction Catalase performs a very elegant 'reshuffling' of toxic compounds.  In the following peroxidative reaction,  a second family of reactions catalysed by Catalase, possibilities for the compound RH2 include phenols, formic acid, formaldehyde and alcohols: H2O2 + RH2   to   2H2O + R, The trick of taking toxins and the potentially harmful H2O2 and recombining them to produce harmless or useful products and water seemed to me to be  a very neat one. Unfortunately details are scarce. I have not yet found details of Catalase's specificity for different RH2, or how readily it catalyses peroxidative reactions as compared to catalysing hydrogen peroxide disproportionation.

Some Processes Producing H2O2 Both reactions catalysed by Catalase, hydrogen peroxide disproportionation and the peroxidative reaction consume H2O2.  Catalase activity in the cell is therefore important for some of the following processes:

Peroxisomes partially oxidise fatty acids producing H2O2 as a byproduct. This peroxisomal oxidation shortens the fatty acids to length C8 or longer and facilitates an energy efficient degradation in the mitochondrion. The peroxisomal oxidation is slightly less efficient at ATP production than is mitochondrial oxidation. However, it is less of a waste of available energy than it might seem. Some of the 'missing energy' is locked up in the oxidative power of H2O2 which is used in the peroxidative reaction described earlier.

Another redox reaction which indirectly involves Catalase concerns the production of DNA. Riboncleotide reductase is responsible for conversion of ribonucleotide diphospates to their corresponding deoxyribonucleotide diphosphates. Ribonucleotide reductase has a tyrosyl free radical which is essential to its action. This radical is produced by an enzyme, NAD(P)H:flavin oxidoreduxtase, which releases superoxide ion, O-2• and this highly reactive radical is converted to H2O2 by the action of Superoxide Dismutase: 2O-2•  +  2H+   to   H2O2 + O2, The H2O2 so generated is then a substrate for Catalase.

In plants, H2O2 is generated in photorespiration - a process that apparently wastefully undoes some of the work of photosynthesis! Photorespiration occurs through the synthesis of Glycolate from 3PG in the Chloroplast and conversion of Glycolate back to 3PG via the peroxisome.  This uses energy in NADH and ATP, consuming O2 and releasing CO2 in the process. One hypothesis is that photorespiration protects plants from photooxidative damage when light levels are high and there is insufficient CO2.

Catalase - A Bifunctional Enzyme? When I first learned of Catalase, I found it extraordinary that Catalase could have essentially two distinct catalytic roles, each involving H2O2.  Given that the single Fe centre in the prosthetic heme group surely participates in the active site, it suggests that a single active site has two functionalities!  The broad spectrum of specificity for R in the peroxidative reaction is less of a puzzle, since only one Carbon atom of R is involved in bond making and breaking.  Wide variation in the rest of 'R' is possible if it does not need to conform to the surface of Catalase. Looked at as chemical equations, in the peroxidative reaction a molecule such as CH3CH2OH takes the place of one of the H2O2 in the disproportionation reaction.  If the same active site and essentially the same mechanism perform both functions, it would seem that H-O-O-H and H-C-O-H (where H and CH3 are attached at C) must adopt near identical conformations when in contact with Catalase.  If  H-O-O-H fits the active site in Catalase well, one would normally expect H-C-O-H to be a very poor fit. This extraordinary bifunctionality of Catalase led me to wonder whether there is perhaps some as yet unrecognised post translational modification or allosteric effect that specialises and fine-tunes Catalase for one or other of its two roles.  If this were so, we might discover more about it by comparing Catalases in peroxisomes of different tissue types where the relative importance of disproportionation and of  peroxidative reactions should be different.. One might expect that some Catalases are better at the peroxidative reaction than others.  It turns out that this is so. Catalases fall into two main classes, the HPI and HPII Catalases. HPII Catalases catalyse just the disproportionation of H2O2 whereas HPI Catalases have both of the described activities.  This still leaves the question of how HPI Catalases achieve their bifunctionality. HPI Catalases exist as two isozymes, HPI-A and HPI-B and these sediment at slightly different densities. I would be interested to know whether these two isozymes of HPI Catalase correspond to the two distinct functions, whether or not they are coded for by the same gene using alternative intron splicing and how the relative abundance of HPI-A and HPI-B is regulated.

Catalytic Perfection of Catalase Catalase is unusual in another respect. It is a textbook example of enzyme efficiency.  It is claimed that Catalase is at the upper limit of how efficient an enzyme can be.

An important limit to the rate of any enzyme catalysed reaction in solution is the frequency with which enzyme and substrate molecules collide with each other. This diffusion controlled limit is in the range 108 to 109 M-1s-1. In this context Catalase's catalytic efficiency (kcat/KM) of 4.0x108M-1s-1 is very high indeed. Because the efficiency is at the diffusion limit Catalase is said to have achieved 'Catalytic Perfection'. The conventional claim is that the enzyme catalyses a reaction almost every time it encounters a substrate molecule.  [Two other enzymes which are also close to 'Catalytic Perfection' are acetylcholine esterase and fumarase].

Too Good to be True? A reaction on each collision is an incredible claim when you consider that: The active site of an enzyme is only a small fraction of the total surface area of a protein The right relative velocity and orientations of interacting molecules is crucial to their reacting.

In my view the conventional interpretation is too good to be true. It seems more likely to me that some assumption made in calculating Catalase's efficiency relative to the diffusion limit is not valid.   An alternative way to look at this is that Catalase has found a way to overcome the calculated diffusion limit, possibly some way to achieve more collisions, with a lower proportion of collisions leading to a reaction

Overcoming The Diffusion Limit I can see just a few ways in principle that an enzyme of any kind could overcome the diffusion limit: Potential Gradient: If the substrate is charged a potential gradient can bring the molecules together "faster than diffusion". This could work in vivo but not in vitro (in a homogeneous system) for it needs structure for this to work. It's a non-starter for explaining Catalase's efficiency as the substrates are uncharged. Enzyme complexes: The E.Coli pyruvate dehydrogenase / dyhydrolipoyl transacetylase / dihydrolipoyl dehydrogenase enzyme complex is an enzyme complex providing "efficient feed through" of substrates between enzymes. As part of a multi-enzyme complex dihydrolipoyl dehydrogenase 'sees' a higher concentration of dihydrolipoamide than is present in free solution.  Enzyme complexes are a common cellular mechanism for beating the diffusion limit. Cellular Structure: Membrane bound enzymes will encounter molecules that preferentially bind to the membrane at a higher rate than they will in free solution.  This is one example of how higher level cellular structure can be important in overcoming the diffusion limit in vivo.  The existence of a peroxisomal compartment achieves similar benefits to those described under enzyme complexes, for by having H2O2 producing enzymes in the peroxisomal compartment with Catalase, feed through to Catalase is improved.  There is further structure within the peroxisome which may be relevant as may the peroxisome's location in the cell.  In tobacco leaf cells peroxisomes, a paracrystalline core can take up most of the volume.  Typically this core makes contact with a chloroplast along one surface.  In fat storing cotyledon cells g lyoxosomes,(an alternative name for peroxisomes), can be seen making close contact with lipid bodies where the fat is stored.  In both cases one would anticipate that a higher level "efficient feed through" is in operation here with substrates being fed through from the adjacent organelle. All three of the above reasons, 1-3, are non starters for explaining measurements of Catalase's efficiency made in vitro. Additional 'weak' binding sites:  This is the explanation I favour for Catalase's efficiency. Binding sites of relatively low affinity will increase the effective local concentration of a substrate. They therefore act as a "buffer" aiding transfer of the substrate to the active site. They can also affect the relative speed and orientation of substrates. A charged group can act as a weak binding site for polar molecules for it will increase the local concentration of polar molecules - as happens with shells of water molecules around a solvated ion. The diffusion limit still operates but we are now dealing with 'diffusion into a sphere of influence', rather than diffusion to the active site. The increased surface area of the sphere of influence relative to that of the Catalase molecule itself increases the diffusion limit. Although the effect of weak binding sites is easiest to describe in terms of a single Catalase molecule in isolation, at least in principle weak binding sites could help Catalase activity in a co-operative process.  If Catalase enzymes interact with each other, they could in principle collect and orient Hydrogen Peroxide molecules for each other. Increased Effective Size: We tend to think of enzymes as compact folded structures, because that is how crystal structures show them.  We tend to forget how dynamic proteins are, and that N or C terminal ends of the chain may be partially unwound. With a weak substrate binding site near an N or C terminal, a protein in a more extended conformation could have a much larger effective surface area, and therefore a greater rate of encountering substrate. An examination of the known structure for Catalase should show whether the C or N terminal ends could be free in this way, and so increase the sphere of influence. Bubble 'catalysis':  (An after thought).  When a dilute aqueous solution of H2O2 is shaken, formation of O2 bubbles is promoted.  This effect is not simply due to the additional energy being added to the system, for slight warming of the solution adds considerably more energy without such a marked increase in bubble formation.  Bubbles in themselves promote O2 release. I don't know whether this effect can rightly be called 'catalysis', or whether it is significant in measurements of rates of Catalase catlysed decomposition of H2O2. In principle it could be a factor amplifying the rate of H2O2 decomposition in vitro and so making Catalase appear closer to the diffusion limit than it is.  If so it would also be interesting to examine the peroxisomal structure described earlier to see whether its structure could enhance the effect, e.g by keeping bubble surface area high.