Protein kinases and protein phosphatases phosphorylate and dephosphorylate a plethora of proteins. They are responsible for regulating the majority of cellular activities. Because of their importance, they can seem intimidating to tackle as a research project. At the end of the day however, kinases and phosphatases are- simply put- enzymes. Therefore, you can standard enzyme assays to study them.
So, 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 suffice it to say, 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 interest of providing a bit of background, however, I’ll attempt to provide some key points about enzymes here.
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. For example:
- An enzyme participates in acid-base catalysis: Will a chemical reaction only take place with a protonation or deprotonation step? No problem! An enzyme might have acidic side chains like aspartic acid or glutamic acid to donate protons to a substrate, and then take them back. Alternatively, the enzyme might contain lysine or arginine to scoop up protons..
- An enzyme participates in nucleophilic substitution: Often, in the reaction mechanism in which 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!
- 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+). They can also be 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.
- Even when not directly participating in the reaction chemistry, the enzyme interacts with the substrates and products. Enzymes stabilize developing negative charges through electrostatic interaction with metal cations, positive charges from lysines, and hydrogen bonds with aspartic acid. Similarly, they can stabilize developing positive charges with glutamate and dipole interactions with the carbonyl oxygen of asparagine and other chemical groups.
- Group hug: overall, what an enzyme does is bind and orient 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!
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. A catalyst also cannot shift 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 can an enzyme convert two substrates into products if their normal equilibrium should be all the way to the left.
Why Is an Enzyme Special?
I often use the following metaphor when describing the role of enzymes in metabolism and physiology:
Imagine a series of pipes leading left and right, up and down, with water flowing in every direction. Imagine also, that there are valves intermittently dispersed throughout these pipes.
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.
What it can do is cut 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 again, the flow is restored, provided the water is still present.
In much the same way, enzymes can control the flow of biochemical intermediaries between metabolic pathways. Enzymes throw their own kinetic parameters (valves) into the interesting mix of physiology, like their maximal catalyzed rate of reaction (Vmax) or their half-saturation constants (Km or S0.5). They can be shut down or activated by small chemical groups directly added onto their scaffolding. They can be activated or inhibited by competitive or non-competitive effectors. In short, they are much more complex catalysts than their chemical counterparts.
This is just a first glimpse in to the world of enzymes. In coming articles, we’ll examine some of the considerations that you must take into account once you’re ready to get your hands dirty and work with enzymes. We will also discuss different ways to study enzymes. This includes the kinetic spectrophotometric assay to look at enzyme activity as well as binding assays such as Förster Resonance Energy Transfer (FRET) and surface plasmon resonance SPR technology.