"The enzyme molecule" Part 1 (Some of the basics)

This post will probably take me further than you need to go (hence Part 1!), but since my original interest in Science really came from my first tutorial on enzymes, as an undergraduate at Sheffield with the late Bill Ferdinand, whose book cover is shown on the left, you'll just have to forgive me! I was explaining the significance of catalysis yesterday to a young Molecular Biologist, and it reminded me of how Molecular Biologists think quite differently than Biochemists. Before I get onto the details consider the relative differences in molecular weights of a typical enzyme and a typical substrate. A molecule like glucose has a Molecular Weight of approximately 200Da. The enzyme Glucose oxidase (for example) has a MW of 80 000Da (and it often comes as a dimer, but we generally think of enzymes per active site). So, you would need 200g of glucose to make one litre of a 1M solution, but 80kg of GOD (not a bad abbreviation for an enzyme?). That's a 400-fold difference simply with respect to mass. It isn't too surprising then to find that enzymes are in much lower concentrations than substrates in the cell. In fact the enhancement of a chemical reaction rate by an enzyme, in some respects is reflected by this ratio: it is a rule of thumb that the ratio of substrate to enzyme concentration, in a typical reaction will be often more than 10,000:1 and we find by experience that the concentrations of biological molecules found in cells approximates as follows:

   Substrates   0.1-10mM
   Coenzymes    0.1-10μM
   Enzymes      0.1-10nM

If an enzyme is going to exert any impact on the rate of a reaction therefore, there must be an explanation for this significant difference in molecular weight and by implication, molecular size (the image on the RHS is of aspartate transcarbamoylase, a large, multi-subunit enzyme with both catalytic and regulatory sites). One other point that is not lost on the producers of proteins, is that the relatively small amounts of enzyme needed for research needs means that they often manufacture and ship nano gram quantities in very small volumes (PCR enzymes often come in 10μl samples, and cost £200!). On the other hand, the level of insulin needed by sufferers for therapeutic reasons can be several grams per patient per week. Insulin itself isn't an enzyme of course, but nevertheless it is, I think helpful to think of these metrics in the context of enzymes.

Some key definitions: an important part of any area of study. An enzyme is a biological molecule that enhances the rate of a chemical reaction, whilst remaining unchanged at the end of the complete catalytic cycle. Most enzymes in living organisms are polymers of amino acids, which we call proteins; but some RNA biopolymers have catalytic activity. RNA enzymes, or ribozymes are much less common in vivo, but occupy several important niches and are important from a Biotechnology perspective. We will not cover ribozymes in the core material of this Unit, but their discovery around 30 years ago has influenced our thinking on the origins of catalysis during the emergence of Life on Earth!

Enzymes enhance reaction rates by lowering the energetic barrier to product formation: we refer to this as the "activation energy" (exemplified on the LHS by the hill (barrier) and the young chap (the enzyme): the ball is both substrate and product. I should add here that the ball at the top of the hill is called the transition state: it is neither substrate or product and is chemically highly unstable in solution (but not in the active site of an enzyme). Enzymes lower this barrier, using a range of different strategies. These strategies may be deployed in different ways and to different extents by different classes of enzymes. However, the tricks employed by enzymes include:

High affinity recognition of the substrates (note this will be qualified in respect of the transition state affinity later on during the unit). We often refer to the affinity of an enzyme for its substrate as the Km (Michaelis constant, which has units of molarity): what would you expect the Km of GOD to be for glucose? 

Orientation of chemically reactive groups, derived mainly from the side chains of amino acids) in the vicinity of the target bonds to be broken or formed. (Think of constellations of amino acid side chains surrounding the portion of the substrates that are to be chemically altered). The diagram on the right shows the catalytic triad found in many enzymes that hydrolyse other proteins (serine proteases). What side chains do you think will be deployed in the reaction mechanisms of typical enzymes and why?

Enzymes sometimes harness the reactivity of cofactors and coenzymes to increase the rate of a reaction. Can you think of how cofactors such as metal ions or molecules such as NAD(H) fulfil this role?

When an enzyme has catalysed the chemical stages of a reaction, it is often the release of one or more products that prevents an enzyme from moving on to another substrate molecule: we refer to this as product inhibition. Can you explain why this sometimes happens and why information of this kind is often helpful to pharmacologists?

Some enzymes follow Michaelis-Menten kinetics, that is their rate of reaction is largely determined by the rate of access of the substrate(s) to the active site and the subsequent release of product(s). However, some enzymes can be modulated or regulated, by the addition of a non-substrate small molecule (or metabolite). We refer to such enzymes as allosteric enzymes. Can you think why such a phenomenon has evolved and how it might be used fruitfully in a metabolic pathway?

With all of these observations in hand, we should now be able to explore how enzymes are able to achieve massive rate enhancements, under mild conditions: essentially neutral pH, medium ionic strength and at relatively low temperatures. You might like to think why proteins seem to be more efficient than nucleic acids as enzymes? Why enzymes are so much higher in mass than their substrates (mainly)? What proportion of a typical genome encodes enzymes? How you might go about designing a drug to inhibit an enzyme? 

Part 2 and beyond will cover the various classes of enzymes with examples of how they work. I will discuss the relationship between the three dimensional structure of an enzyme and its activity and the mechanisms of enzyme regulation again with examples. I will also explore the evidence for all of these ideas and concepts, as well as the limitations of our knowledge!