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Introduction

A Rational Approach to the Study of Biochemical Pathways

 

The most difficult task for most students of biochemistry is making sense of,and learning, the various pathways. There are so many of them, and each one has its own constellation of reactants and products, enzymes, cofactors, reaction mechanisms and regulatory or control mechanisms. It soon becomes apparent that, at some level in a given organism, these pathways are interrelated either through common intermediates or feedback control mechanisms. How does one even begin to learn this complex subject, let alone complete the task of integrating all of the information into a coherent understanding?

As with most things, itís probably best to start at the beginning. We all learn better if we recognize the underlying principles and commonly repeated themes or motifs in a discipline of study. As we proceed through our lectures, I will point these out, in an attempt to apply such generalizations to the specific tasks at hand. On the other hand, some reactions have a beauty that resides in their uniqueness, and often in their simplicity, and we need to appreciate these also. I am sure that your background has already provided you with many of the principles that we will discuss, and, if so, this will serve as a review for you.

How can one make sense out of a series of biochemical reactions that comprise a pathway? I suggest the following approach:

(1) Look at the entire sequence as a whole to put it into perspective. How many steps are there? Is this a pathway that you have heard about before?

(2) Next, look at the individual steps. Read the names of the reactants, products , enzymes and cofactors involved. Some of these will be familiar from other pathways, so you might begin thinking about how these pathways are related. See if you can determine the names of reactants and products from the molecular structures. As the names usually follow systematic rules, this practice will reinforce these rules and make it easier to identify them. Practice writing the names and their corresponding structures. Learning biochemical pathways is an active process. I donít think that there are many of us who can just look at a reaction scheme and passively absorb and remember it. Look at the name of the enzyme that catalyzes each step. Can you tell what the name of the enzyme is, just by looking at the reactants, products and type of reaction?

(3) Try to identify any cofactors involved. These are the small organic molecules (coenzymes) or metal ions that are required for the activity of an enzyme. It turns out that most of the metal cofactors are transition metal ions, and they have very interesting chemistries.

(4) Look for steps involving ATP (or GTP). You will recognize that such steps have substantial free energy (DG') exchanges.

(5) Try to identify individual steps that occur close to equilibrium, as well as those that occur far from equilibrium. It is one or more of the latter that are important control points in the pathway.

Enzyme Classification

The Nomenclature Committee of the International Union of Biochemistry and Molecular Biology recommended, in 1992, the following classification of biological enzymes based upon the type of chemical reaction catalyzed:

(1) Oxidoreductases : These enzymes catalyze oxidation-reduction reaction and coenzymes are usually also involved. You will see them written as "reductases" (dehydrogenases), "oxidases" and oxygenases.

(2) Transferases : These catalyze the transfer of functional groups from one molecule to another. They may be called "transferases" but we will often see them as "kinases", in which case a phosphate group is transferred and as phosphorylases, where a phosphate or pyrophosphate is transferred across glycosyl or related linkages.

(3) Hydrolases: These catalyze hydrolysis reactions, in which a water molecule is involved in breaking a bond. These include phosphatases, peptidases and ATPase.

(4) Lyases: In reactions catalyzed by lyases, either a carbon-carbon double bond is generated or something is added to a C-C double bond of a substrate. Decarboxylases, dehydratases and aldolase are some examples. "Synthases" are usually lyases.

(5) Isomerases: As expected, these enzymes catalyze reactions in which a substrate is changed from one isomeric form to another. Epimerases and racemases are examples.

(6) Ligases: In these reactions, two molecules are joined, usually requiring the presence of a nucleotide (like ATP).Synthetases and carboxylases are examples.                                                                                        

You will not be surprised to recognize that all of these enzymes catalyze reactions in which covalent chemical bonds are made or broken. We traditionally think of an enzyme as a molecule which increases the rate of a chemical reaction by increasing the accessibility of a "transition state" between reactants and products, and it does this by decreasing the amount of free energy needed for the reactants to reach the transition state.

That this is an arbitrary restriction on the scope of enzyme activity is commented upon by Daniel L. Purich (TIBS Vol.26 No.7 July 2001). There are many "physical" reactions in which one can identify product-like and substrate-like states that are non-covalent and that are speeded up by biological catalysts, some traditional and some not. Included among these are chaperonin-mediated reactions encountered in some cases of protein folding, chromatin condensation, the operation of molecular motors, active and carrier-mediated transport and DNA processing by polymerases. Many of these physical reactions also require nucleotides to power them. Membrane transporters ("pumps") are now recognized as a special class of enzymes.

Purich argues that most of the ATPases and GTPases are misclassified as hydrolases and, rather, should be a separate class of enzymes called "energases" in which the energy of the covalent bond is converted into mechanical work. Since so much of the remainder of the course will deal with nucleotides, it makes sense to consider this in more depth.

When ATP is hydrolysed, a covalent bond is broken with the result that an inorganic phosphate molecule is liberated (Pi) and ADP remains, and considerable energy is released as heat. The ATPase enzymes fit neatly into the "hydrolase" classification. When the hydrolysis reaction is coupled to the synthesis of a molecule by the addition of two other molecules, the enzyme involved in the synthesis reaction is a "synthetase". Synthetases fit best under the above classification as ligases, and the reactions are chemical because covalent bonds are made or broken. But what about reactions in which a conformational change occurs that is driven by the free energy released by the hydrolysis of ATP? These are really important reactions and we will study one of them when we study DNA polymerase. When an enzyme mediates nucleotide hydrolysis and couples the free energy released to a conformational state change in a system, Purich suggests that the name "energase" be used.

 

We will often run across enzymes that have names that are related to the substrate involved or to the catalytic action of the enzyme involved, and that are not systematic in the sense of the classification above. The reasons for this are historical. There will even be instances in which the enzyme name is unrelated to either. In all such cases, it is instructive to try to classify the enzyme systematically anyway, so as to improve clarity of thinking. The recipe for the systematic name is:

Substrate Name + Major Group Classification + "ase"

In general, though, we will see and use the trivial, rather than the systematic, name.

Finally, it is necessary to remain flexible in our thinking in order to allow for changes in classification, as described for the case of a mechanochemical reaction

Our lectures will start with amino acid synthesis, as you have just finished studying amino acid catabolism. We will then study  the integration and regulation of fuel metabolism and the organ specialization involved in these processes. This will be a good opportunity to pull together what you have learned about all of the major pathways involved in energy production and storage. We will also touch upon insulin signaling , which will  supplement what you have already learned about intracellular communication pathways. Next, we'll look at nucleotide metabolism and after that the lectures will focus on DNA structure, DNA topology,  and DNA-protein interactions that regulate  transcription of the information encoded in DNA. Finally, there will be two lectures devoted to the molecular biochemistry of photosynthesis, particularly the light reaction.

 

Wherever possible, I will relate new information to material already covered. You will be able to follow hyperlinks that will accomplish this also. Finally, biochemistry is not simply a collection of random facts, although it may sometimes seem that way. There are a number of basic chemical principles that, if understood, help to organize the material in this discipline. And ultimately, the biochemistry of an organism is a complex puzzle of mutually interrelated processes, some of whose pieces are still missing. The challenge is to see the whole as being more than the sum of the parts, even though all the parts aren't available.

 

I recommend that you read the sections specified in my course outline prior to attending class (or immediately after, at the latest). The text is "Biochemistry", 3rd  Edition, by Voet & Voet (2004). This material is often supplemented in my notes by other sources, and these will be listed. You can read the original source material if you like but, if you attend class, you will hear me lecture on the material and then you can read my notes. When you read your text, keep the following in mind: your textbook can serve as a reference book for you for many years into the future, so you might as well become as familiar with it as you can. I still refer to texts that I used 30 years ago because I know where to quickly find the information in them that I need.  When you study pathways or unfamiliar molecules, draw them out with a pen or pencil and write out the names. Your brain learns best when you use it in a multimodal fashion.

What about memorization? Contrary to popular belief, I strongly recommend selected memorization as a way to master a subject. But it's pointless to memorize something without first understanding it, so go for the understanding first. There are certain pathways that every self-respecting biochemist should know by memory. The glycolytic pathway and the TCA cycle are two that come to mind immediately. I will let you know when I think that something is worth memorizing and I'll leave it up to you to make the final decision.

 

The first set of lectures will cover the biosynthesis of amino acids, as you have just learned about their degradation.

 

Link Here To See Power Point Presentation For Introductory Lecture MOLECULAR BIOCHEMISTRY II INTRODUCTORY LECTURE