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The Ultimate Antioxidation and Detoxification Machine: Catalase

Catalase is one of the most efficient enzymes known, with reaction rates approaching
200,000 catalytic events/second/subunit, which is near the diffusion-controlled limit. Keep
in mind that the rate of any enzyme-mediated reaction is determined by the frequency with
which enzymes and substrates collide. It is calculated that the enzyme catalyzes a reaction
almost every time it collides with a substrate molecule. Some even claims that catalase is
at the upper limit of how efficient an enzyme can be. Two other enzymes that are nearly as
efficient are fumarase (TCA cycle) and acetylcholine esterase (degradation of
acetylcholine).

So what is the significance of catalase behaving like the ultimate enzyme machine? Well,
take a look at some of its substrates: hydrogen peroxide, alcohol, phenols, formic acid,
and formaldehyde. Get the picture? These substrates are pretty nasty chemicals that can
make your life miserable. Take hydrogen peroxide, for instance, it is a byproduct of
aerobic metabolism normally produced by the mitochondrial electron transport chain.
Excess hydrogen peroxide can damage nucleic acids, proteins, and lipids, causing cell
injury or death, meaning that catalase has an important physiological role in protecting the
cell. It turns out that intracellular hydrogen peroxide is predominantly generated in an
organelle called peroxisome, which is present in all eukaryotic cells. In addition, activated
phagocytes are the major contributor of extracellular hydrogen peroxide.

Several peroxisomal enzymes use molecular oxygen to remove hydrogen atoms from
specific organic substances in an oxidative reaction, thus producing hydrogen peroxide
(RH2 + O2 => R + H2O2). Catalase, being an peroxisomal enzyme, then utilizes hydrogen
peroxide to oxidize alcohol and other organic compounds in a “peroxidative” reaction
(H2O2 + R’H2 => 2H2O + R’). When H2O2 accumulates in the cell, the enzyme acts solely
on this reactive oxygen species by breaking it down to water and molecular oxygen
(2H2O2 => 2H2O + O2). Another enzyme that also utilizes H2O2 as substrate is
glutathione peroxidase, also an important anti-oxidation defense system. Liver, kidney,
and erythrocytes contain the highest amounts of catalase. Interestingly and importantly, a
recent study by Schriner et al. has shown that the life span of rodents could be extended
by overexpression of catalase in mitochondria. This finding not only confirms the long-held
belief that aging is mainly caused by reactive oxygen species, it also offers a potential
molecular strategy through which humans might more effectively fight against oxidative
stress.

Although catalase activity can be directly measured in the ultraviolet region, this method is
not applicable to monitoring cellular catalase activity due to UV absorption by protein and
other molecules present in biological samples. We have developed a unique and simple
colorimetric method to directly assay catalase activity in crude cell extracts. Since the rate
of decomposition of hydrogen peroxide to water and oxygen is proportional to catalase
activity, the first step of the assay system, which is carried out at 37°C for 30 min, is
designed to consume a fixed amount of hydrogen peroxide by catalase. The second step
is then designed to assay for the amount of unconsumed hydrogen peroxide based on
hydrogen peroxide-mediated oxidation of an azo chromogen, which is carried out at 50°C
for another 30 min. Oxidation of the azo chromogen yields a yellowish color measurable by
spectroscopy or more conveniently by a microplate reader. Thus, you’ll be able to
determine in 1 hour how potently your cells might fight against oxidative stress via this
ultimate enzyme machine.

Biomedical Research Service