Bitesize Bio readers who have an interest in enzymology will hopefully remember my recent series on the Basics of Protein Phosphorylation. On the back of that series comes something which is, believe it or not, even more basic- and with perfect timing too, seeing as how Bitesize Bio is launching a webinar series on Chemistry for Biologists.

Protein kinases and protein phosphatases, as I’ve described, phosphorylate and dephosphorylate a plethora of other proteins. At the end of the day, however, kinases and phosphatases are- simply put- enzymes. The proteins that are acted upon by kinases and phosphatases may- or may not- themselves be enzymes.

Our common denominator? Enzymes! With that, we’ll be taking a multi-part look into some of the general concepts, do’s and don’t’s, technical tips and tricks, and potential panic buttons surrounding enzymology. We now begin the first part of “So You Want to Work With Enzymes.”

What is an enzyme?

The long answer is… well suffices to say that there are countless large, thick textbooks out there covering all the details. It’s impossible to condense something like that into a bite-sized article, so we won’t!

The short- in my opinion, much too short- answer is: to think of enzymes as big, biological, glorified catalysts. The problem is that this description scarcely does them justice at all. Why? Well, a chemical catalyst can be defined as something that increases the rate of a reaction, without itself being used up in the process. An enzyme, however, is so much more.

In the interests of providing a bit of background, however, I’ll attempt to provide some key points about enzymes here, in the first part of our series

Proteins- and let’s not forget that at the end of the day, enzymes are proteins with catalytic abilities- are made up of a library of 20 natural amino acids. The order and combination of the amino acids in a protein’s primary sequence is what dictates its structure from the secondary level and up. Within the overall three-dimensional structure of an enzyme, some of the amino acid side chains will face inward, towards the core of the enzyme, where the chemistry of enzyme catalysis takes place.

What does an enzyme do?

An enzyme allows biological chemistry to take place in a variety of ways, often involving the direct participation of the enzyme’s catalytic side chains.

1.      An enzyme participates in acid-base catalysis: Will a chemical reaction only take place with a protonation or deprotonation step? No problem! Acidic side chains like aspartic acid or glutamic acid can donate protons to a substrate, and then take them back. Or, protons can alternatively be scooped up by a lysine or arginine that has a lone pair available.

2.      An enzyme participates in nucleophilic substitution: Often, in the reaction mechanism where one substrate will nucleophilically attack another substrate, it must wait its turn until the enzyme attacks first. A serine or cysteine makes a perfect nucleophile, especially if it can be deprotonated by an adjacent amino acid to a seryl or cystyl anion. Once serine or cysteine is bound to one of the substrates, the second substrate can come along, attack, and toss the side chain along- nothing makes a better leaving group than a whole protein!

3.      Besides the amino acids with which they are synthesized, enzymes often have other little goodies that have been added onto them post-translationally: these are known as prosthetic groups. They can be something simple like cations- most commonly zinc (Zn²⁺) or magnesium (Mg²⁺), or something more complex like thiamine pyrophosphate, an active form of Vitamin B₁, or flavin adenine dinucleotide (FAD), an electron carrying cofactor involved in biological redox reactions.

4.      Even when not directly participating in the reaction chemistry, the enzyme interacts with the substrates and products. Developing negative charges can be stabilized by electrostatic interaction with metal cations, positive charges from lysines, and hydrogen bonds with aspartic acid. Developing positive charges are stabilized by electrostatic interactions with glutamate and dipole interactions with the carbonyl oxygen of asparagine and other chemical groups.

5.      Group hug: overall, what an enzyme does is binds and orients the substrates of a biochemical reaction. Regardless of whether any side chains directly participate in the chemistry, or which cofactors or ions may be involved, it brings substrates into close proximity and correct spatial orientation to allow them to react with one another.

What does an enzyme NOT do?

This, right here, is where our analogy of an enzyme being a catalyst comes into play!

1. A catalyst can lower the activation energy of a reaction, but it cannot change the overall energetics of a reaction. If a reaction is endothermic versus exothermic, or exergonic versus endergonic, those properties cannot be altered by the addition of a catalyst.
2. Tightly linked with the energetics of a reaction is the equilibrium of a reaction. If the equilibrium should be shifted far to a left, a catalyst will not drive it to the right.

So too do these rules hold for an enzyme. If ATP is not available for an ATP-dependent reaction, then the reaction will not happen even with all the enzyme in the world. Nor will all the enzyme in the world convert two substrates into products if their normal equilibrium should be all the way to the left.

Why is an enzyme special? How does it differ from a chemical catalyst? Why is it of so much interest in biological sciences?

I often use the following metaphor when describing the role of enzymes in metabolism and physiology: imagine a series of waterworks, pipes leading left and right, up and down, with water flowing in every direction. Imagine also, intermittently throughout these pipes, are valves.

The valves can’t change the direction of the water flow. They can’t make water that was flowing left to right suddenly flow from right to left, or make water that was falling downward suddenly defy gravity and start climbing upward in the pipes- you’d need a pump for that. And if water is moving at only a trickle, a valve simply can’t make the water start gushing. It can only allow as much water to flow in a direction as it normally would.

But…!

What it can do, is cuts off the flow of water if you close the valve. It can redirect water that was normally flowing along one pathway to another pathway. And if that valve is opened once again, provided that water is still present and trying to force its way through, the flow is restored.

In much the same way, enzymes can control the flow of biochemical intermediaries between metabolic pathways. Enzymes throw their own kinetic parameters into the interesting mix of physiology, like their maximal catalyzed rate of reaction (V_max) or their half-saturation constants (K_m or S_0.5). They can be shut down or activated by small chemical groups directly added onto their scaffolding, like phosphate through protein phosphorylation. They can be activated or inhibited by competitive or non-competitive effectors. In short, they are much more complex catalysts than their chemical counterparts.

In our next part, we’ll examine some of the considerations that must be taken into account once we’re ready to get our hands dirty and work with enzymes- especially if we’re looking to work with a specific type of enzyme.