Quantum Evolution, Johnjoe McFadden. Chapter 5

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Quantum Evolution - Chapter 5 - Life’s actions

’… Enzymes as mousetraps

Enzymes are one of the key ingredients of life and are ubiquitous in living tissue. Moisten a hunk of fresh bread with warm water and then leave it for about ten minutes. Pop the same amount of bread in your mouth and chew it for a minute before taking it out leaving alongside the other un-chewed bread. After the ten minutes is up, taste the unadulterated bread and the bread that that you have already chewed (it is not so disgusting – it was in your mouth just a few minutes before!). The first (just wetted with water) will taste exactly like soggy bread. However, the bread that has been in your mouth will now taste sweet. The sweetness is due the presence of sugars released from starch in the bread by the action of the enzyme amylase in your saliva.

Starch is a polymer of glucose that is manufactured by plant cells as an energy store for their seeds. It will hydrolyse (react with water) spontaneously to release glucose sugar, but the reaction is normally very slow. Enzymes are catalysts that speed the rate of chemical reactions by directing the motion of electrons and protons along specific paths. The enzyme in our saliva that breaks down starch is called amylase. Chewing bread impregnates it with amylase and so stimulates the breakdown of starch more than one million-fold.

Your body has the capacity to make thousands of distinct enzymes each with a specific role to accelerate at least one of the millions of chemical reactions taking place in your body every day. To discover how enzymes work, we will examine one of them in more detail. If like me you are relatively unfit, then you will experience breathlessness, tiredness and possibly even cramp as you run the length of a football pitch. This discomfort and weariness is due to oxygen starvation in your muscles. When muscle cells have plenty of oxygen they burn glucose completely to carbon dioxide (this metabolic burning is called respiration and will be discussed more fully below) and the energy released is used to drive muscle contraction. However a plentiful supply of oxygen is required for respiration. During vigorous exercise the blood supply cannot keep up with the oxygen demand so glucose is only incompletely broken down to a chemical called lactic acid. The muscle weakness and cramp you might suffer during vigorous exercise are due to the build-up of this lactic acid in your muscles. The breakdown of glucose to lactic acid involves a chain of chemical reactions; each accelerated by its own enzyme. The last enzyme in the chain, lactate dehydrogenase or LDH, converts pyruvate to lactic acid. Like most biochemical reactions, it proceeds extremely slowly in the absence of a catalyst. LDH accelerates the rate of the reaction more than one billion fold.

As with all chemical reactions, the reaction catalysed by LDH involves the migration of electrons; in this case the transfer of a pair of electrons from a molecule called NADH to pyruvate to yield lactic acid. Enzymatic reactions that involve donation or removal of a pair of electrons usually involve the molecule NADH, which acts as an electron carrier in the cell. After donating its pair of negatively charged electrons NADH becomes the positively charged ion, NAD⁺.

We know a great deal about the reaction catalysed by LDH because the enzyme has been crystallised. The crystallisation process aligns all of the protein molecules in the same direction within the crystal. In the technique called X-ray crystallography, X-rays can then be shone through the crystal to obtain a kind of X-ray of the molecule. Scientists who study this X-ray pattern can map the positions of each of the amino acids and charged groups within the protein to discover how the enzyme works.

The enzyme can be very schematically represented as a mousetrap. The substrates (NADH and pyruvate) float into a cavity within the enzyme called the active site. A positively charged amino acid (arginine), at position 171 in the protein (171^st amino acid from the left end of protein) serves to anchor the lactic acid; and another arginine at position 101, serves to anchor the NADH. These anchoring groups act like the trigger of a mousetrap to release a sequence of shape changes within LDH that folds the protein over the substrates and swings an armoury of charged amino acids to attack their chemical bonds. The carbonyl bond (C=O) of pyruvate is very electronegative and vulnerable to attack. A positively charged histidine amino acid at position 195 of the enzyme delivers the coup de grâce, by spitting a proton into this carbonyl group to form a new covalent bond, the hydroxyl (O-H) group of lactic acid. The extra positive charge (from the proton) acquired by the substrate sets up a wave of electron migration throughout the molecule that draws a pair of electrons plus a proton from the NADH cofactor, to yield the products, NAD⁺ and lactic acid.

After the LDH enzyme has performed its job on pyruvate, it plucks a proton from a water molecule to replace the one that it lost. This returns the enzyme to its starting state and readies it to attack another pyruvate molecule. It is a very important principle of all enzymology that the enzyme (or indeed any catalyst) must emerge unscathed from its chemical gymnastics. Only in this way could a single molecule of enzyme convert millions of molecules of substrate into product. It is this facility to be reused that makes enzymes so effective as biological catalysts.

As you can see, the key to the action of LDH, and indeed all enzymes, is the directed movement of protons and electrons within the active site of the enzyme. The dynamic interaction between the substrates and enzyme directs electrons and protons along the paths that lead to the products. Enzymes perform directed actions.

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Animals obtain our energy from food. Food serves as both as a source of energy and as a source of raw material from which to construct new cells or repair old ones. We are what we eat. Let us return to your football game at the point where you are about to sprint into action. The millions of myosin molecules in your calf and thigh muscles are ready to initiate their cycle of grasping and releasing the actin filaments but a crisis soon ensues – they are out of ATP! You cannot run along to the local store to obtain and guzzle down a jar of ATP, like Popeye’s can of spinach, because you cannot run without ATP. Instead your body must break down glucose (or other sugars) from your food to make ATP. Although the glucose molecule holds plenty of chemical energy, it cannot be used directly for muscle contraction. Instead muscle cells must transfer their incoming glucose supplies into the more readily utilisable form of ATP. Glucose contains six covalently bonded carbon atoms tied together in a ring structure. With a plentiful supply of oxygen, cells are able to completely oxidise one molecule of glucose to yield just carbon dioxide, water and about 30 molecules of ATP.

Which brings us to another vitally important chemical reaction: oxidation. Oxidation is what goes on when you burn paper, wood or glucose in air and it involves electron motion. Electrons in the relatively high potential energy rooms (orbitals) in the atoms of paper, wood or glucose, travel down a potential energy gradient to fill the vacant low potential energy electron rooms in oxygen. The difference in potential energy is emitted as the light and heat of a fire. The energy released from burning wood can be harnessed to make things move: steam engines power the wheels of a train and in the process, chemical potential energy is converted into kinetic energy. Cells similarly burn metabolic fuels in an oxidation reaction that yields chemical energy in the form of ATP. The reaction is known as respiration and, just like burning, it usually requires oxygen. The efficiency of our cellular engines is pretty good, about 38% of the total energy available from glucose oxidation is captured into ATP. The rest is dissipated, mostly as heat; which is why vigorous exercise makes you hot.

Respiration takes place inside cellular organelles (the organs of cells – structures that do specific tasks) known as mitochondria. The structure of mitochondria is the first indication of the remarkable nature of respiration. They have many of the features of whole cells, including internal membranes, ribosomes and even their own DNA. Mitochondria are even able to divide independently from the rest of the cell. It is now thought that, like plant chloroplasts, mitochondria are the direct descendants of symbiotic bacteria.

In respiratory oxidation, the electrons from glucose are first dumped onto the NAD electron carrier molecule that I mentioned above, to form NADH. The electrons in NADH - still at high potential energy – are then plugged into mitochondria, where they are passed - like a relay baton - through a series of enzymes that carry them down the potential energy gradient towards oxygen. Mitochondria are bounded by a pair of membranes - one inside the other - with a water-filled space in-between; and it is within these membranes that all the electron transport action takes place. Respiratory enzymes, inserted into the inner membrane, act as electron-powered proton pumps to capture protons from the inside of the mitochondria and transport them into the water-filled space between the membranes. One of the proton-pumping stations is an enzyme called cytochrome oxidase, whose position in the electron relay is to receive electrons from another enzyme called cytochrome c, and pass them to oxygen. Buried within cytochrome oxidase are three atoms of copper, which pluck the electrons from an iron atom within the cytochrome c enzyme and then push them on to oxygen. The enzyme is inserted into the inner membrane so that one end of the protein faces towards the mitochondrial matrix on the inside of the mitochondrion, and the other end of the protein faces the intermembrane space. On receiving electrons the enzyme undergoes a shape change that causes a charged amino acid on the matrix side to pluck a proton from a water molecule within the matrix. A second shape change causes another of its charged amino acids to spit a proton into the intermembrane space. The net result is an electron flow-driven pumping of positively charged protons from one side of the inner membrane to the other, to generate an electrical charge difference across the membrane of about 0.15 Volts – in other words, a battery.

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The mitochondrial battery is then used to power ATP synthesis. The enzyme involved is called ATP synthase or ATPase and it acts as a molecular motor. The enzyme spans the inner mitochondrial membrane so that it experiences the proton gradient generated by the respiratory enzymes. The enzyme faces more than ten times the number of protons at its head end (pointing into the intermembrane space) than its tail end (pointing into the mitochondrial matrix). A channel in the protein allows protons to flow through the protein. This proton flow causes the enzyme to rotate - rather like the rotation of a turbine engine or a water wheel. Just as the energy of a rotating water wheel can be harnessed to grind corn, so the energy of the rotating protein can be harnessed to synthesise ATP. The enzyme rotates in steps of 120 degrees and each step is associated with the synthesis of a single molecule of ATP.

The precise mechanism by which the rotation of ATP synthetase is coupled to ATP synthesis is still unclear. A reasonable conjecture would be that the rotation twists the protein structure that, like a coiled spring, can be made to do work. Quite likely the shape changes within the protein bring about charge migrations that make ATP. However, such a remarkable enzyme may have even more surprises up its sleeve so nobody is betting on the precise mechanisms yet.

In any case, we know enough about respiratory ATP synthesis to be certain that it is one of the most remarkable phenomena in the known universe. We marvel at the engineering wonders of our modern world – the jet aircraft, the Channel Tunnel, the Millennium Dome, the roaring Godzilla doll; but these pale into the commonplace beside the marvellous action of those tiny electric-powered proton pumps and proton turbine engines that power every living cell within your body every day.