"The enzyme molecule" Part 2 (Practical enzymology)

Glutamate Dehydrogenase: an enzyme at the heart of energy metabolism.

Introduction. The enzyme glutamate dehydrogenase (pictured RHS) is ubiquitous in eukaryotes; forming a link between energy production via the Krebs (or Tricarboxylic Acid [TCA] Cycle. Early investigations described the substrates and products, but it was between 1960 and 1990 that most of the molecular work on this enzyme was published. The pioneering enzymological work was carried out on the enzyme from bovine liver (beef liver) and in particular the laboratories of Carl Frieden (now at the University of Washington in St. Louis) and Paul Engel (now at University College Dublin); more recently a great deal of crystallography has been carried out on bacterial GDHs in the laboratories of David Rice and Pat Baker at the University of Sheffield. The first GDH to be crystallised was isolated from an anaerobic bacterium, Clostridium symbiosum at Sheffield in the late 1980s by Engel, Rice and Hornby. This work provided a framework for understanding the catalytic mechanism of this enzyme and has been followed more recently by the determination of structures for vertebrate GDHs, in 2002.

The reaction catalysed by GDH (sometimes abbreviated to GluDH or GLDH), lies in the direction of ammonia assimilation. However, the equilibrium constant makes it possible to measure both directions, and in this project we shall mainly utilise the oxidative deamination of glutamate for our measurements.

Glutamate +NAD(P)+  ↔ 2-oxoglutarate + NH₄⁺ + NADH + H⁺

Our understanding of the conversion of foods such as carbohydrates (eg glucose), lipids (fats) and proteins was one of the major achievements of 20^th century Biochemistry and Physiology. Later developments in our understanding of the concepts of “building blocks” and the biosynthesis of our own body fats, carbohydrates and proteins helped us to appreciate the complex systems that cells, tissues and organisms employ to not only produce energy, but also to grow and develop. The recent term “Systems Biology” is used by many to describe the integrated sets of reactions and pathways that define the function of an organism at a range of hierarchical levels.

For example, you might be interested in ATP synthesis. This is the product of oxidative phosphorylation in the mitochondria of vertebrates. However, ATP is also produced as a product of a large number of enzyme catalysed reactions. Moreover, ATP is utilised in the biosynthesis of glycogen, the synthesis of nucleic acids and is a regulator of many biochemical activities (the transfer of the terminal phosphate of ATP to certain proteins can selectively turn on (or off) their catalytic function.  This suggests that studying individual enzyme catalysed reactions, while important, has to be seen in a wider context. This is probably the most important aspect of “Systems Biology”. GDH catalyses the mobilisation of 2-oxoglutarate (also called α-ketoglutarate) either to generate the amino acid L-glutamate for incorporation into newly synthesised proteins, or to provide a valuable metabolite for neuronal signalling. In the reverse reaction, the enzyme “tops up” the TCA cycle (see top RHS), since the reaction catalysed by 2-oxoglutarate dehydrogenase precedes the formation of succinyl CoA.

The key information for today's lab class is below. Further details will be discussed during the session: this is just to give you an idea of planning your experiments. You must obtain all of the necessary reagents and equipment as a group from the usual locations in Innovation Lab 1.

Practical enzymology with GDH. In order to fully appreciate how enzyme's catalyse reactions, we need to observe them in action. We are going to begin with preparing the reagents for measuring the conversion of NAD to NADH via the oxidative deamination of glutamate, catalysed by Clostridium symbiosum GDH. The measurements of rates will be discussed in the next blog in some detail, but first I want to discuss the preparations in advance of rate measurements.

The substrates. The substrates are provided as solid sodium salts of glutamate and nicotinamide adenine dinucleotide (NAD+). Both were obtained from Thermo Fisher. You should make notes on the details provided by the manufacturer, including any chemical characteristics, molecular weight and notes on storage requirements.

Each group should prepare 100ml of 200mM glutamate in distilled water. You should measure the pH and aliquot the solution into 10x10ml Falcon tubes for storage.

Similarly, you should prepare 10ml of 10mM NAD+ and store 10x1ml Eppendorfs. No need to measure the pH.

Make sure your samples are clearly labelled and that you have stored the solutions correctly.

The buffer. Prepare a 5x concentrate of PBS and check its final pH. (we shall use lower and higher pH buffers later for measuring the influence of pH on reaction rates.

Standard reaction mixture:

50mM glutamate
1mMNAD+
PBS
Final volume 1ml
Enzyme volume 5ul
Room temperature

The enzyme. You are provided with a suspension of GDH in saturated ammonium sulphate at a concentration of 20mg/ml. The activity of the enzyme preparation must be established by making a series of dilutions. We will use PBS for diluting the enzyme samples, we will then establish the appropriate enzyme dilution for measuring rates and end points reproducibly.  

The aim of this week's lab work is to "see enzymes in action"; having demonstrated this, in a reproducible manner, we can begin to investigate how temperature, pH, inhibitors such as heavy metals (which tend to be non-specific) and competitive inhibitors (similar to drugs), influence reaction rates in vitro. These experiments will reinforce the concepts that we have discussed in the seminars. Additional information is available in Unit 13 at the Google Classroom.

"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!

The Biochemical Basis of Energy generation from everyday food

In defining Biochemistry (previous Blog), one of the most important research questions that occupied the minds of the pioneers of the subject was how organisms extract energy from foods, such as carbohydrates, fats and proteins. Through a series of elegant experiments that occupied Biochemists during the first half of the 20th century, it became clear that complex carbohydrates (for example) are broken down through a series of enzyme catalysed steps, into a series of intermediates, leading ultimately to the production of ATP (top LHS), the universal energy currency in most living organisms (I say most because there are always exceptions!). I like to think of ATP generation more as a recycling reaction, in keeping with the theme of the First Law of Thermodynamics; which describes the conservation of energy in the Universe. Understanding the properties of ATP and similar biological nucleotides is an important aspect of Biochemistry and you can find a nice description of the chemistry and nomenclature of nucleotides at this link, provided by Dr. Scott Bellos an MD and former Chemistry Graduate at Rensselaer Polytechnic Institute in New York,  whose learning resources in Nucleotide Biochemistry are highly recommended. 

The question I always ask myself, when I read about the central role played by ATP in biological energy transactions, is why not GTP, or CTP etc. The first point to consider is that the release of energy as the phosphate ester bond is cleaved generates approximately 30kJ/mol of energy (usually expressed as ΔG^o' = -30.5 kJ/mol for ATP hydrolysis to ADP and P_i). [I shall comment on units in a separate studentmicroblog]. Is this the same for GTP etc? The answer is of course yes, so there is nothing special about this particular nucleotide triphosphate. The preference for ATP in Biology, is probably just a chance, evolutionary event, in which adenine was fixed as the primary component of many biological cofactors and metabolites, including NAD, NADP, FAD, acetyl CoA etc. [Look them up on Google Images and make sure you can see the adenine moiety]. The evolution of adenine binding pockets in proteins was an important, early discovery in the development of X-ray Crystallography of proteins and has reinforced the priority position of adenine, over guanine etc in Biochemistry. The work of Michael Rossmann and John Walker, among others, was important in understanding the elements of primary and tertiary structure in protein recognition of adenine and its various derivatives (see image top RHS).

There are several factors that help us understand why the use of molecules like ATP has evolved to provide the fuel for many of the enzymatic steps that underpin metabolism.

1. ATP can be hydrolysed to release phosphate (Pi) yielding approximately 30kJ/mol of energy that can in turn be coupled to otherwise unfavourable reactions. In fact, it is not uncommon to find two hydrolysis steps (ATP to AMP) contributing 45kJ/mol more energy to an even more challenging biochemical reaction (the example that springs to my mind is the attachment of amino acids to their cognate transfer RNAs in protein biosynthesis). The work of Fritz Lipmann is of central importance in our appreciation of ATP in Bioenergetics. The ATP entry in Wikipedia is well worth an hour of your time on a Sunday morning!

2. The release of energy from the above hydrolysis reaction is a consequence of the relative "ease" with which ATP breaks down to ADP in water. It is important to realise that there is no "magical" packet of energy in ATP, but rather, in the aqueous, cellular environment at a pH slightly above neutrality, the equilibrium position of ATP=ADP +Pi, lies in favour of ADP, since the favourable interactions between water (Hydrogen bonds etc) and ADP are more stable than the covalently bonded phosphate. The repulsion between the phosphates in ATP, which is partly stabilised by Mg2+ ions, is also less entropically favoured over the hydrolysed products (ADP + Pi). The most important point to note is that ATP hydrolysis liberates sufficient energy to drive most of the thermodynamically unfavourable reactions in Biology. Occasionally ATP is hydrolysed to AMP to provide a "double shot" of energy and sometimes, special measures must be employed (which you will discover at University level). The evolution of metabolism has therefore been considerably constrained by the physical chemical properties of ATP and this is why it is such an important molecule for we Biochemists to understand. As an exercise, make a list of 10 reactions that incorporate ATP in living organisms.

3. Think of ATP as a rechargeable battery. At any one time, each individual probably "carries" 25g of ATP in their cells. However, it has been shown that each of us recycles our own body weight in ATP every day! We now need to consider how the food we eat generates ATP and where in the cell this takes place. A point made clear by a landmark publication in the late 1950s by Hans Krebs and Hans Kornberg (whose names you will find on the Innovation Lab bench and who [a few years before the above picture was taken) used to be the head of the Sheffield students union!), where they set out very elegantly, the underlying principles of energy metabolism in living cells. 

4. It is important here to note that the bulk of ATP is generated through (at the time) an unexpected mechanism. Unlike the enzymatic transfer of one chemical group (eg a phosphate) to an acceptor (eg an amino acid side chain on a protein), ATP in eukaryotes is synthesised through a sequence of events that involves the flow of electrons and the transfer of protons across the inner mitochondrial membrane. This electron transfer chain of molecules that are essential for this to take place is complex, highly organised and together with the ATP synthase (see my Molecule of the Month) results in the generation of 3 ATP molecules from every NADH liberated by the Krebs Cycle (or 2 ATPs from FADH). This will be discussed in detail in the classes.

We shall use the classic "Boehringer Mannheim" Metabolic Map, first produced by Gerhard Michal in 1965 (so we are celebrating its 50th Birthday this year!), which I think came out of the earlier Nicholson maps in the late 1950s, to illustrate the networks and inter-relationships between metabolic pathways. Think of metabolism as the London Underground, or the Paris Metro. The important points about metabolic pathways is that they are rarely free-standing and they comprise a series of enzyme catalysed reactions, some of which are regulated. [The mechanisms of regulation are diverse and can include chemical modification of the enzyme, controlled hydrolysis or synthesis to regulate the "amount" of enzyme [I shall discuss steady state phenomena in a separate Blog]. It was also discovered in the 1960s that some enzymes can be "modulated" by small molecules that are distinct from the substrates or products: we refer to these as allosteric effectors and they can either activate or inhibit an enzyme catalysed reaction]. Importantly, the conversion of molecules like complex carbohydrates takes place via a series of reactions, some of which decompose and some modify the intermediates. The purpose of these pathways is to generate energy (ATP) or to recycle the carbohydrate into the molecules that make up the organism. This includes other metabolites, other carbohydrates, fats, proteins etc. This is why an appreciation of metabolism is such a defining part of Biochemistry. Moreover, the malfunctioning of metabolism and its regulation lie at the heart of many diseases and is critically important in drug discovery, which will be discussed to illuminate several aspects of metabolic principles.

I shall discuss enzyme catalysis in the next post.

Key points

Energy in living organisms is stored and released in the form of ATP

The hydrolysis of ATP is harnessed to unfavourable biochemical reactions (this is called coupling)

Foods like carbohydrates and fats are "processed" by all living organisms by a set of enzyme catalysed reactions to generate NADH and FADH, both of which provide the route to ATP synthesis on the inner mitochondrial membrane.

These pathways are interconnected, complex and serve not only to produce energy, but to build the organism, both structurally (skin and bones, or leaves and stems) and functionally (enzymes and genes).

The metabolic pathways that were charted in the last century are generally found in all living organisms (aerobic and anaerobic organisms do have significant differences), but there are some interesting and instructive exceptions.

The regulation of metabolism by a range of Biochemical strategies is an important phenomenon that has become a key aspect of drug discovery since it is often faulty in a number of diseases.

What is Biochemistry? A taster for Unit 13

We shall shortly begin Unit 13, entitled Biochemistry and Biochemical Techniques. I thought it would be useful to define the subject areas and perhaps the best place to begin with is the Biochemical Society, founded in 1911 and influenced heavily by the first Department of Biochemistry established in Liverpool under the leadership of the first Johnston Chair of Biochemistry, Professor Benjamin Moore FRS. Sure enough, the website is bristling with exciting information via many links, but no definition! I then looked at the journals that specialize in Biochemistry. The Biochemical Journal, founded in 1906, alas, no joy there either! I wondered if Biochemistry, the equivalent US journal might help us out. Well, a little:

Biochemistry publishes research from the arena where biochemistry, biophysical chemistry, and molecular biology meet. The journal covers structure, function, and regulation of biologically active molecules; gene structure & expression; biochemical mechanisms; protein biosynthesis; protein folding; global protein analysis and function; membrane structure-function relationships; biochemical methods; bioenergetics; bioinformatics, and immunochemistry.

But, for me this is too vague and "understandably" inclusive of related fields, but it doesn't give me a concise definition, rather the scope of that particular learned journal. The Journal of Biological Chemistry is another famous American journal that has published many landmark papers in Biochemistry over the last one hundred years (110 to be precise!). It's mission is defined on their web site as:

The Journal of Biological Chemistry publishes papers based on original research that are judged to make a novel and important contribution to understanding the molecular and cellular basis of biological processes.

So, some themes are emerging, but no precise definition. The journal Biochemistry published since 1936 (as Biokhimiya) from Moscow is similarly vague, as is the Japanese Journal of Biochemistry, launched in the 1920s by the Japanese Biochemical Society.

So inevitably, I went to Wikipedia! here I find the following definition:

"Biochemistry, sometimes called biological chemistry, is the study of chemical processes within and relating to living organisms. By controlling information flow through biochemical signalling and the flow of chemical energy through metabolism, biochemical processes give rise to the complexity of life. Over the last 40 years, biochemistry has become so successful at explaining living processes that now almost all areas of the life sciences from botany to medicine are engaged in biochemical research.Today, the main focus of pure biochemistry is in understanding how biological molecules give rise to the processes that occur within living cells, which in turn relates greatly to the study and understanding of whole organisms."

And it's pretty good! I might have written it a little differently, but the essence is there. Let's see what the BTEC Unit 13 expects of us:

"The aim of this unit is to develop the learners knowledge and techniques needed for the study of biochemistry. Learners will investigate biological molecules, enzymes, metabolic pathways and the structure of proteins."

This was the primary of focus of Biochemistry and Biochemists from the late 19th century (the "Father of Biochemistry", Otto Warburg is pictured above: note the similarities with the Innovation Labs) until the mid 1960s. Of course questions remain unresolved in some of these areas today, but the body of knowledge accumulated comes largely from this period. And 100 years of Biochemistry is a lot to capture in one Unit! However, we are going to make a start!

Let me begin with a plan, that fits the aims:

1. Biomolecules. We shall consider the range of molecules found in living organisms, the conservation of such molecular species between all forms of life (as well as some areas of specialisation) and the chemistry and physics associated with their biological roles.

2. Enzymes. What are they, what do they do and what do they look like? We will use glutamate dehydrogenase as our model enzyme and investigate its catalytic properties, its structure and mechanism, together with the influence of factors such as heat, ionic strength, pH and inhibitors on its activity. We shall also compare GDHs from bacteria (including C.difficile), fungi, plants and animals, with a particular focus on mammalian forms of the enzyme. We shall develop our understanding through a series of lab sessions, culminating in the determination of the Vmax and Km of the enzyme from Clostridium symbiosum and a demonstration of how these parameters are affected by a competitive inhibitor.

3. Metabolic pathways. The pioneering work of Nobel Laureates including Otto Warburg and Hans Krebs laid the foundations for our understanding of contemporary metabolism. This is essential for understanding nutrition and human (as well as all organisms) physiology in health and disease. The flux of metabolites through glycolysis, beta oxidation and the Krebs Cycle, leads ultimately to the generation of ATP via the organised electron transfer chain in the mitochondrial membrane of eukaryotes. The enzyme glutamate dehydrogenase provides a link to the biosynthesis of proteins and together we shall assemble a map that provides us with a detailed understanding of the role of metabolic pathways, enzymes and the consequences of environmental stimuli and nutritional status. We shall finally contextualise our understanding  of metabolism in the contemporary field of Systems Biology.

4. The structure of proteins. The phrase "structure and function" fills the pages of most Biochemistry text books. We shall use examples such as antibodies, enzymes and gene activators (RHS) and repressors, to explore this topic. This will be intimately linked to my Molecule of the Month Blogs and we shall host academics who have solved structures of key proteins to illustrate the importance of this area of Biochemistry.

I hope this has whetted your appetite for more!

Welcome to the first post!

This is the first post on my latest Blog site, dedicated to support students taking the BTEC route(s) in Sciences at the Liverpool Life Sciences UTC. However, it is an open Blog and anyone is welcome to look, read, comment and collaborate. The purpose of the Blog is primarily to support the Unit based material with an emphasis on the Innovation Lab experimental programme, taken by students in Y12 and 13. In addition to specific posts that will relate to experiments coming up, or results just behind us, I intend to pepper the Blog with related aspects of Science (with an emphasis on the Life Sciences) that I think are interesting. However, any suggestions are also most welcome and I shall develop the page format as the term develops and time permits. You will also see links on the Right Hand Side (RHS) to my other Blog sites, some of which will be unrelated, but some will be relevant. I sometimes duplicate posts if I think they may be useful to the different, intended audiences.

So what should you look forward to? The BTEC Level 3 programmes at the UTC in Liverpool present students with a parallel portfolio of science and health related education opportunities, offering an alternative to A levels. The Innovation Labs provide a laboratory based focus for the Science Units and we shall be running Innovation sessions relating to Applied Science Units 13, 15 and 18 (in the first instance).

Unit 13: Biochemistry and Biochemical Techniques
Unit 15: Microbiological Techniques
Unit 18: Genetics and Genetic Engineering

The first post this week, will precede the Unit 13 programme (commencing 19th January), which is being developed in conjunction with the Department of Molecular Biology and Biotechnology at the University of Sheffield and one or more of our UTC Partners. This will centre on the enzyme glutamate dehydrogenase (LHS), but will incorporate all of the elements required by Unit 13. The posts will be an accompaniment to materials and resources provided through Mr. Houseman's Google Classroom at the UTC, for which students will have secure access.

More soon....