--- Catalase ---
......The primary reaction catalyzed by catalase is the decomposition of H2O2, to form water and oxygen.
2 H2O2 --> O2 + 2 H2O
...... Were aliens to land on earth and take off their space suits, they would probably gasp and die from the oxygen in our atmosphere. ..Oxygen is, of course, a strong oxidizer. ..Furthermore, most metabolism in the presence of atmospheric oxygen leads to the production of hydrogen peroxide. ..It is the peroxide that kills. .. Earth-life has figured out two ways of getting around this toxic problem of "peroxide." ..One is not to make it. ..The other is to have an enzyme that destroys it. ..The bacteria that are in cheese use the first strategy. ..Almost all of the rest of us make catalase, which we use to split peroxide into a water molecule and an oxygen atom.
...... "QUALITATIVE" EXPERIMENTŠŠ Take a few drops of the suspension that is marked yeast, and add it to a milliliter of 10% hydrogen peroxide. ..Note the fizzing! That's the oxygen gas split from the peroxide by the enzyme. .. Does your saliva contain catalase? .. (Don't be a dummy: do NOT put the peroxide solution in your mouth; put some spit in a tube and add the peroxide!) ..How about your partner's blood? (Yes, it does! - so you don't need to assault your partner!) // "40 volume" hydrogen peroxide (=20%) can be purchased from your nearest beauty supply store.
Now let's have a little fun with CATALASE!
...... On the lab bench you will find a wide-ranging collection of various biological items - many of them you will recognize from the supermarket: lettuce, potatoes, beans, corn, yeast, hamburger, mushrooms, Swiss cheese, cheddar cheese, onions, oysters or clams, fern, moss, pine and maple or oak. .. Sort these out according to their kingdoms (for cheeses, think of the "dairy bacteria" rather than cows!). .. Then within the plants, sort them according higher or lower taxa, and even noting which are monocots and which are dicots.
...... Each group should collect a small piece - maybe only 1 cm2, and then place it in the bottom of a small test tube and mash it with an aluminum stirring rod or blunted aluminum nail (do NOT use IRON or STEEL!). .. Add a few drops of 1.5% H2O2 to the tube, swirl, and then take note of which fizz and which don't. .. Some will fizz violently, others very slowly. ..The question is not so much quantitative, but qualitative. .. If it fizzes, it must have catalase, and if it has catalase then it must have the gene encoding that enzyme's structure. .. We want to know if we can duplicate the easy work that led to so many Nobel Prizes, as you will see in the next "inspirational" section. Will the fizz test work in our hands?
* * * * *
...... You should now have a feel for what enzymes are. .. "So what good is that?" some wag in the back row mumbles. ..Good question, so let's take a more philosophical look:
INSPIRATIONAL OVERVIEW ("catalase")
...... Before we become immersed in the technologies of enzymological analysis, let us look at the science, the wonder-why-things-are-the-way-they-are stuff. Science is the art of exploration, the art of asking good questions. ..Technology is figuring out how to answer the questions and, hopefully, then to do it quantitatively. ..A scientist is one who unlocks a door to a room into which many technicians flood to inventory the contents. ..It took Watson, Crick and Wilkins to reveal the IMPLICATIONS of the structure of DNA, and then it took literally thousands of researchers to check them out to see that molecular biology really did work that way.
...... Let us now turn our wondering minds to how we "burn" our calories without being on fire. ..You know it's by means of enzymes, which number in the thousands of different types, each with its own specific ability. ..You also know that enzymes are a class of catalysts. .. Let's take a look at the discovery of one of the earliest chemical catalysts. ..It was known that the gases hydrogen and oxygen could be mixed together 2:1 in a container, and that they would sit there quietly for as long as you wanted to stare at the container. ..But just expose the mixture to the smallest bit of cold metallic platinum. BOOM! ..Well-, now that you have picked yourself up from the other side of the room, you have learned what a catalyst is by traumatic experience! ..Supposing that you could find all the remains of the reaction, you would still have your speck of platinum metal, and a drop or two of water. ..The important thing: the catalyst was unchanged - that's a major part of the meaning of catalyst.
...... Of course we don't "burn" our calories that explosive way. And therein lies the beauty and the wonder! ..Our catalysts - our enzymes - are able to work together on both constructive (anabolism) and destructive (catabolism) activities to make us what we are in an overall process called metabolism.
...... One of the great biochemical physiologists of our time used this southern California analogy for describing enzymes to his students. ..The water-rich areas of northern California could supply the arid regions of southern California with sufficient water IF ONLY the water could economically be transported south to Los Angeles and San Diego. ..The problem was that while the rivers of northern California were two thousand feet higher than Los Angeles (free energy), there was a huge barrier of more than 7,000 feet between them. ..To lift the millions of gallons of water needed each day over the mountains was NOT an economical idea (the activation energy needed was too expensive!). ..However, one man had an idea (like DNA has ideas encoded in it). ..He passed his idea on to other engineers (mRNA?), who then went out and trained thousands of workers (enzymes!) who were to bore tunnels through the mountains so that now gravity alone was sufficient to move the water into the Los Angeles basin. ..However, the workers refused to work unless they were paid (a type of activation energy!), and unless they had the right kinds of machinery and supplies (other types of activation energy and enzymatic cofactors). ..Thus after expending several years of hard labor and millions of dollars on materials (the total activation energy expended by the workers), the water flowed. ..That waterway still flows after many decades. ..The amount of activation energy expended to bore those tunnels is much, MUCH less than lifting all that water over the Sierra Nevada and Coastal Mountain Range.
...... But this physiologist was quick to say that these are not completely analogies: ..the free energy drop, the activations energies are not analogies: ..they are exactly that in physics terms rather than in chemical terms. ..Remember, that these are thermodynamic laws that pertain to all chemistry, all physics - well, everyTHING in the universe!
...... Let us take a Watson-Crick-Wilkins approach in the investigation of one particular enzyme that is very popular among living organisms. .. Remember to investigate the common things, for therein most probably lie universal laws.
The IMPLICATIONS of the fizzing caused by placing drops of hydrogen peroxide on living organisms.
Let us step back in time. Imagine being the discoverer of an enzyme. ..Because you are a trained observer and can recognize the world-shaking importance of what you see, you become very excited with the results of this simple hydrogen peroxide fizz test. .. Visions of gold Nobel Prizes dance in your head. ..Let's follow your train of thought so that you may become a role model for young aspiring scientists.
...... You found "fizz" activity in almost every LIVING thing you inspected except in a few bacteria, which neatly separated themselves into two groups - in those which were found plentifully in dairy products, and in those which could not be grown in the presence of air (oxygen). ..The former group were eventually to be called the "Lactic Acid Bacteria" or more commonly the 'dairy bacteria.' ..These can grow in the presence of air, although they do not use it. ..And the latter group consists of what became to be called the "strict anaerobes," because they cannot grow in the presence of toxic oxygen. ..So you have three groups of life - (a) everything in four Kingdoms, and most of the millions of types of bacteria, (b) the twenty of so species of dairy bacteria, and (c) the hundred or so species of strict anaerobes - as in toxic shock, gangrene, and botulism.
...... You speculated that this data must mean that there exists some pathway, that produces H2O2, since a catalyst to break it down would not likely be there were H2O2 not often present. .. So you scribbled out a pathway, which if unbalanced and far from complete would look like this:
Food ---> ..?.. ---> ..?.. ---> H2O2---> H2O + O2 (fizz!)
...... "And that's it!" you'd excitedly exclaim. .."Let me clarify it for my lab partner." ..Since whatever it was that catalyzed the conversion of H2O2 into fizz, you cleverly called "catalase," using your discoverer's prerogative to name new things.
...... But where did the H2O2 come from in the first place. .. "Probably by reducing some oxygen, and that means that some electrons had to be added to it by some other catalyst we'll call Enzyme-1:
Food ---> ..?.. ---> (? + 2e- + O2) --(Enz1)-> H2O2--(Catalase)--> H2O + O2
...... Now here is where your tremendous powers of observation kicked in. ..Because almost all life has catalase, they must also have Enzyme-1, otherwise there would be no H2O2 and thus catalase wouldn't have been needed to detoxify the body of this highly oxidative toxin. ..And since Enzyme-1 probably uses oxygen as a substrate, maybe you are getting a glimpse of the biochemical basis of respiration! ..(Oh, can you hear the tinkling of golden Nobel medallions!)
......"Ah HA!" you shout. .. "That's why the three groups of life!" you reasoned.
......Group (a), above, has both enzymes: enzyme-1 and catalase.
......Group (b) has neither and therefore cannot make H2O2 and hence doesn't need catalase for detoxification as it grows in the presence of air.
......Group (c) has Enzyme-1 but does not have catalase, so that if it is exposed to air during growth it makes lethal H2O2, which it cannot detoxify, and the cells die. ..Hence, these few organisms can only grow in the complete absence of air.
...... And because the reduction of O2 to H2O2 involves the transfer of electrons from who-knows-where, perhaps this will involve something like our body's hemoglobin. ..Let's look for hemes, which are in many respects like chlorophyll.
...... "Well, what do you know," your partner informs you. .."All but the dairy bacteria have heme-like compounds in them. ..Since they are colored and in most cells, let's call them 'cytochromes.'"
...... So you see that this mere little fizz-test leads to the discovery of the electron transport pathway, and that to oxidative phosphorylation. ..Prospects of Nobel Prizes now glitter very brightly in your eyes!!!
...... BUT, you are not finished. ..There is yet more to be revealed, more to be discovered!
...... An old tenet of evolutionary phylogeny decrees: Simplicity begets Complexity. ..The dairy bacteria are the simplest - they lack both cytochromes and catalase (another heme enzyme, by the way!). .. Maybe the dairy bacteria came first - they had not yet figured out how to make the heme structure for either Enzyme-1 or catalase. ..Then came the strict anaerobes onto the stage of life. ..They had figured out how to make the heme structure and how to use it to make Enzyme-1, but because they had evolved into an early world devoid of oxygen they had no worry of making H2O2 because any electrons produced would go to reduce something else that was not toxic. ..Finally, with the advent of simple cyanobacterial photosynthesizers (aka bluegreen 'algae') and their production of oxygen, which escaped into the atmosphere, a detoxifier was needed and came into being - catalase! ..Because H2O2 is so rapidly lethal this new enzyme had to be even more efficient than "very good." ..Catalase is one of the fastest and most efficient enzymes known. ..A native, membrane-bound catalase molecule can detoxify nearly 100 million H2O2 molecules per SECOND! .. (Isolated "purified" catalase is only about 10% as fast.)
...... So now our little fizz-test reveals evidence supporting such notions as 'early earth atmosphere', and evolution in the most fundamental early episodes of life on earth. ..So you enroll in a weight lifting course just so that you will be able to carry all the gold Nobel medallions!
...... Wisdom is learned: It may be that the simplest tests might hold the greatest lessons. ..Almost all Nobel Prizes were won for work that was done simply and in a short time. It is not the amount of labor one puts in, it is the thought and observation that wins the day!
...... The Prizes came not from tons of data, but just for unlocking and cracking open a previously unknown door in the universe. ..You peeked in. .."WOW!" you exclaimed as you saw all the implications. ..Soon herds of technicians (which are most of today's scientists) rushed in to collect the tons of data. ..The new realm became of scientific vogue. ..You, the discoverer, at first were considered a wacky odd-ball, but as the mounting technical data confirms what you thought, you became famous and the weight training came in handy.* ..You were the real scientist who scratched your head and rubbed your chin, and wondered why this bit of the universe was the way it was.
...... Fizz -> an enzyme -> respiration -> early evolution of life on the planet! ..Wow!!! ..This, dear students, is what science is about. ..That's the "SO WHAT?" ..And you have seen it all - and working in your own two hands!
* Just a few Nobels on this line of thought: Buchner (cell-free fermentation); Krebs and Kornberg, Embden, Meyerhof and Parnas; Korkes and Gunsalus. ..And once electron transport and oxidative phosphorylation were mostly elucidated, that opened the doors to the Nobels for photosynthesis.
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