Hello again my Biochem people,
We have arrived at week 10, only three weeks and counting until end of term. I think that I am more excited than sad. This week has been rather calm even though next week brings with it our DREADED IN-COURSE MOCK EXAM and we should be reviewing all the weeks work. Maybe it was ‘the calm before the storm’. Week 10 was focussed on tutorials, class quizzes and TCA/ETC as well as the two online review quizzes.
A few comments on each of those items. Let me begin with the review quizzes. I was a bit hesitant to attempt these quizzes without having studied previously and recollect all the finer details of cells, carbs and proteins. Therefore, looked over my notes and studied for the day before attempting the quiz. I was slightly disappointed when I did the quiz and the questions were quite straightforward and simple. I felt that the quiz could have tested more in-depth understanding and application of knowledge on the material. On the glycolysis quiz, I was even more surprised that the questions were all the questions from the Glycolysis MCQ worksheet YouTube video that Sir put up for us to test our knowledge of the important aspects of glycolysis. I must say though, unlike the cells quiz, I was glad that the glycolysis quiz was based on information that I already knew. Moreover, do not get me wrong, I am by no means complaining. Nevertheless, I thought that these quizzes were meant as a refresher to aid in studying for the mock exam and therefore would be slightly more challenging. I must say though, that if this is a reflection on the type of questions being asked, I am not as nervous about next week’s mock exam.
Tutorial focussed on enzyme kinetics and glycolysis. Very enjoyable session, at least the most enjoyable thus far. Sir not only asked some tough questions that forced us to apply all the knowledge that we knew on glycolysis, he also guided us as to the most appropriate means of answering essay questions. This session was also a good review of the enzymes and assimilating all the information we have on enzymes and proteins into developing an appropriate answer for questions. This method, I believe was the ultimate goal for tutorials, for us to take all the information that we are learning and apply it not only to questions of specific subject matter, but also to related topics and real life situations.
As far as lectures are concerned, week 10 was very light. Even though it continued into TCA and ETC, there were little details and main focus was on the general overview of both processes. I believe that the focus was on getting ready for the mock exam. Those two quizzes in class were really helpful. It taught me two very important things: even though you know, the answer you need to write it down quickly and accurately and DOESN’T UNDERESTIMATE SIR WHEN HE SAYS BE PREPARED FOR A QUIZ AT ANY DAY. I must admit that I did browse over the information for class, but I was not as sharp as I wanted to be.
I wanted to talk a little about something that seems to be cropping up often and that is enzyme-inhibitor kinetics. Even though there were many examples in class, I feel it necessary to go over the basics in each category.
We will begin with inhibitors. Inhibitors are substances that alter the rate of reaction of an enzyme-catalysed reaction. There are two main classes of inhibitors: IRREVERSIBLE and REVERSIBLE.
Irreversible inhibitors: these are substances that bind to the active site of the enzyme and form a covalent bond between the R-groups of the active site and the R-groups of the inhibitor. As you know, during enzyme-substrate binding, the formation of a weak bond occurs to allow for easy dissociation and release of the products and substrate from the active sites of the enzyme. However, with irreversible inhibitors, there is the formation of a strong covalent bond that causes a decrease in the activity of the enzyme. This bonding prevents the substrate from binding since the active site is pre-occupied and the strong bonding of the inhibitor prevents the detaching and hence renders the enzyme inactive.
Irreversible inhibitors may also be called suicide substrates. Why you might ask? These substances are similar to the substrate of the particular enzyme i.e. substrate analogues. These analogues become highly reactive and activated by binding to the active site of the enzyme or near the active site and cause the formation of covalent bonds between the active site and the substrate analogue. This then inactivates the enzyme.
Most irreversible inhibitors are natural or synthetic toxins that exhibit competitive, irreversible inhibition. Examples of these may include:
Cyanide: now you are wondering where in world I would get cyanide from unless someone is blatantly trying to poison me. The answer is quite simple, bitter almonds may contain cyanide. Cyanide, reacts with metal ions in the enzyme structure such as iron, zinc and copper and form irreversible bonding that alters the action of the enzyme. Enzymes found in the electron transport chain are highly vulnerable to this and may cause decreased energy production.
Sarin: this is also called nerve gas and it binds to the serine residues found in the active sites of enzymes to form that covalent bond that renders the enzyme inactivated. The enzyme most vulnerable is acetylcholinesterase which is responsible for the breakdown of acetylcholine, a neurotransmitter, from the synaptic cleft of neurones. Acetylcholinesterase in effect regulates the action of acetylcholine by decreasing the activity on the postsynaptic membrane. However, if acetylcholinesterase is inhibited it allows for the prolonged action of acetylcholine on the postsynaptic neurones. Acetylcholine is responsible for motor activity in the body as well as wakefulness and learning. Therefore, increased activity of this neurotransmitter causes the constant contraction of muscles and thus may alter ability to breathe since respiratory muscles are hypertonic and unable to function properly. This may lead to the individual dying of asphyxia.
Penicillin: this reacts on bacterial cell walls. Penicillin binds to the active site of the enzyme, glycopeptide transpeptidase. It forms a covalent bond with the serine residues present in the active site to render the enzyme inactive. Glycopeptide transpeptidase is responsible for the crosslinking of the peptidoglycan cell wall in gram-negative bacterial. Inactivation of the enzyme causes weakening of the bacterial cell wall and thus increases vulnerability to cell death. Weakened cell walls also decrease bacterial growth and thus reduce infections caused by bacteria.
There are many more examples of irreversible inhibitors, but the main idea is the loss of function of the enzyme leading to dire consequences for the organism.
Reversible inhibitors on the other hand form non-covalent bonding with the enzyme and therefore, this inhibition may be reversed or overcome. It is a temporary state. Reversible inhibition may be sub-classified into four main categories: competitive, non-competitive, uncompetitive and mixed inhibition.
Competitive inhibition: the substance or compound is similar to or closely resembles the substrate for the particular enzyme. The inhibitor competes with the substrate to bind to the active site of the enzyme. Once the inhibitor is bound to the active site, it forms a non-covalent bond with the active site and prevents the substrate from binding to the enzyme since the active site is already occupied. This leads to a decrease in the affinity of the enzyme to bind to the substrate. This decreased in affinity may show an increase in the Km value for the enzyme since with the increase in the concentration of the inhibitor, there is increased binding of the inhibitor to the active site of the enzyme due to out-competition of the inhibitor to the substrate.
However, this inhibition may be overcome by an increase in the substrate concentration. This increase in substrate concentration facilitates the out-competing of the substrate over the inhibitor and so the substrate will bind to the enzyme more than the inhibitor. Hence, the maximum velocity or rate of reaction of the enzyme is unaffected by this inhibition. Therefore, Vmax is unaffected.
This inhibition may be observed graphically via M-M curve and L-B plots.
M-M curve showing the effect of competitive inhibition on an enzyme. There is an increase in Km and no effect on Vmax.
L-B plot for competitive inhibition.
A classic example of competitive inhibition is observed in the metabolism of ETHANOL and the drug, DISULFIRAM/ANTABUSE. Alcoholic beverages possess ethanol, which is metabolized in the liver by two enzymes. Firstly, ethanol is oxidized to acetaldehyde by the enzyme alcohol dehydrogenase in the cytosol and then rapidly to acetic acid by the aldehyde dehydrogenase in the mitochondria of hepatocytes. Therefore, there is little accumulation of acetaldehyde in the body. Acetaldehyde is responsible for the side effects or hangover effects felt by alcohol consumption such as nausea, vomiting, headaches and light-sensitivity. Hence, quick metabolism of this substance is necessary.
Disulfiram is a drug that is utilized by recovering alcoholics in order to aid in overcoming drinking habits. Disulfiram competes with acetaldehyde in the mitochondria for binding to the enzyme aldehyde dehydrogenase. When disulfiram binds to the active site of aldehyde dehydrogenase, it prevents acetaldehyde from binding and thereby causes an increased accumulation in the cells and lead to the severe side effects.
Ethanol may also be utilized as a treatment or antidote for methanol poisoning by similar mechanisms. Methanol may be oxidized to formaldehyde and formic acid, which are severely toxic to the cells of the body, especially the optic nerve, leading to blindness as well as respiratory and neurological effects. Ethanol may be administered since it competitively inhibits the action of alcohol dehydrogenase enzyme in the liver. When ethanol binds, it decreases the affinity of alcohol dehydrogenase for methanol and thus decreases the oxidation of methanol, since the products of methanol oxidation is toxic to cells rather than methanol itself.
Another type of reversible inhibition is non-competitive inhibition. With non-competitive inhibition, the inhibitor binds to the free enzyme at another site other than the active site of the enzyme or to the enzyme-substrate complex. It does not resemble the substrate and binding to these sites enable a change in conformation of the active site leading a decrease in the formation of products. Thus, by this inhibition, there is a decrease in the maximum velocity of the enzyme and hence a decrease in Vmax. However, it does not affect the substrate from binding to the active site of the enzyme rather the inhibitor prevents the formation of products by altering the interaction of the enzyme and substrate. Therefore, the affinity of the enzyme for the substrate is unaffected and Km is unchanged.
This may be illustrated by M-M curve and L-B plot.
M-M curve showing the effect of non-competitive inhibition. there is a decrease in the Vmax but Km is unchanged.
L-B plot illustrating the effect of non-competitive inhibition.
An example of non-competitive inhibition is ALANINE AND PYRUVATE KINASE. Alanine, the amino acid is formed from the transamination of pyruvate. When there are high amounts of alanine produced, it acts as a negative feedback onto the enzyme pyruvate kinase. As you know, pyruvate kinase catalyses the reaction that dephosphorylates phosphoenolpyruvate to pyruvate in the last step of glycolysis to yield ATP. Increased levels of alanine, causes increased binding of alanine to pyruvate kinase and in some cases to pyruvate kinase-phosphoenolpyruvate complex and prevents the formation of pyruvate. This leads to decrease in formation of pyruvate and regulation of amino acid production in times of plenty.
Uncompetitive inhibition is another class of reversible inhibition. The inhibitor binds ONLY to the enzyme-substrate complex in this process. Binding of the inhibitor prevents the formation of products by causing a change in the interaction between the enzyme and the substrate. Therefore, there is a decrease in the maximum velocity of the enzyme and the rate of reaction of the enzyme and so Vmax is reduced. Since the enzyme-substrate complex exists in equilibrium to the enzyme and substrate, when there is a shift in the equilibrium by binding of the inhibitor to the enzyme-substrate complex, there is a compensation effect. This means that there is increased binding of substrate to free enzyme active sites to attempt to recover the equilibrium by formation of more enzyme-substrate complexes. This gives the appearance of an increase in affinity of the enzyme for the substrate due to increased binding and complex formation. Hence, there is a decrease in the Km for that particular enzyme. Therefore, there is increased use of substrate than in a non-inhibited state. To note with uncompetitive inhibition, there is a proportional decrease in the Vmax and Km values of the enzyme. This means that both are decreased in proportional value due to the equilibrium compensation.
This may be observed graphically on M-M curves and L-B plots.
An example of uncompetitive inhibition involves the drugs, PROBENECID and ZIDOVUDINE. Probenecid is a drug that is utilized to treat chronic gout. Zidovudine is a potent HIV inhibitor that decreases levels of HIV in the blood by inhibiting the replication of HIV. Zidovudine is metabolized in the liver where it undergoes glucuronidation to become a glucuronide that is less reactive to the body. Thus, the liver decreases the activity of zidovudine. It was found however, that probenecid binds to the glucuronide enzyme-zidovudine complex in the liver to decrease the glucuronidation of zidovudine.
The last class of reversible inhibition is mixed inhibition. There is binding of the inhibitor to the free enzyme as well as the enzyme-substrate complex similar to non-competitive inhibition. This leads to a decrease in the rate of reaction of the enzyme and hence a decrease in Vmax, however, there may be a change in the affinity of the enzyme for the substrate and so there may be an increase or decrease in the Km value.
All right then folks, I hope that this information is easy to understand and helps with your studies.
Therefore, to conclude, Week 10 has come and gone and now we are in deep preparation mode for this exam on Wednesday. I am both scared and excited. This one is supposed to test our readiness for the final and give an assessment of the amount of work that we require to do. So all the best to my fellow Biochemians and happy studying.
Bye for now and keep up the struggle.
– Biochemistry by Dr Jakubowski. Chapter 6- Transport and kinetics. Models of enzyme inhibition. 19th March, 2013.
– MyMCAT: start your prep now. Enzyme Inhibition. 11th July, 2010. http://www.mymcat.com/wiki/Enzyme_Inhibition#Competitive
– ChemPages Netorials. Biomolecules: Enzymes. http://www.chem.wisc.edu/deptfiles/genchem/netorial/modules/biomolecules/modules/enzymes/enzyme4.htm
– Pathway detail: Ethanol metabolism. http://flipper.diff.org/app/pathways/info/2980
– Elmhurst College. Virtual Chembook. Enzyme Inhibitors. 2003. http://www.elmhurst.edu/~chm/vchembook/573inhibit.html
– Enzyme Kinetics. http://www.uvm.edu/~mcase/courses/chem205/lecture13.pdf
– Enzyme inhibition. http://www.pearsonhighered.com/mathews/ch11/c11ei.htm
– Kokbiolab. Lecture 7: Enzyme Inhibition. http://www.google.tt/url?sa=t&rct=j&q=&esrc=s&source=web&cd=12&sqi=2&ved=0CIwBEBYwCw&url=http%3A%2F%2Fwww.kokbiolab.com%2Flib%2Fexe%2Ffetch.php%3Fmedia%3Dfnkok%3Alec7-inhibition.ppt&ei=_-xZUePjKdTi4AOiy4GgDw&usg=AFQjCNFr8Q66T9oJ6P9hQda0DnyrBsN09w&sig2=WOO_7GK9PpDG5biHJfe4hA