Model Questions for Biochemistry-III

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 Model Questions for Biochemistry (Microbiology) 
1. Explain in brief about lipolysis.
2. Explain the urea cycle.
3. What is the salvage pathway, enlist the steps?
4. Why TCA is said to be amphibolic?
5. What are the ketone bodies? Explain their metabolism.

6. What are enzymes, explain with the classification?
7. Give some examples of the special molecules of the body whose precursor are amino acids.
8. Enlist the enzymes involved in TCA cycle and estimate the number of ATP generation.
9. Describe the stoichiometry for palmitic acid oxidation.
10. What are the differences between fatty acid biosynthesis and breakdown.

11. Derive Michaelis-Menten equation.
12. What are coenzymes and isozymes? Explain in brief with examples.
13. Explain pyruvate dehydrogenase complex.
14. What is the glyoxylate cycle? Show its relationship to the citric acid cycle.
15. Explain the De novo synthesis of Pyrimidine (steps only).

Salivary amylase and starch

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Salivary amylase is a digestive enzyme secreted by the salivary glands, and about 1.7 liters of saliva is produced everyday by a healthy human. It is responsible for breakdown of the polysaccharide “starch” into dextrins (oligosaccharides), maltose (disaccharide) and glucose (monosaccharide) in the mouth.

Starch consists of two types of molecules, “amylose” (20-30%) and “amylopectin” (70-80%) and both are polymers of glucose. Amylose is a linear chain of glucose units that are linked by α-1,4 bonds, whereas amylopectin is a branched molecule in which glucose units are linked by α-1,4 bonds in the straight chain region in addition to α-1,6 bonds at the branching points. The relative proportions of amylose to amylopectin depend on the source of the starch.

Branch of amylopectin

Salivary amylase hydrolyses only the α-1,4 bonds that link glucose units. Therefore, starch and glycogen, which consist of α-1,4 bonds, are hydrolyzed by amylase while. The cellulose consisting of β-1,4 bonds, cannot be hydrolyzed by salivary amylase. The α -amylase constitute a family of endo-amylases that catalyze the initial hydrolysis of starch into shorter oligosaccharides through the cleavage of α-D-(1-4) glycosidic bonds. Neither terminal glucose residues nor α-1,6-linkages can be cleaved by α-amylase.

The conformation of (α-1→4) linkages in amylose, amylopectin causes these polymers to assume coiled helical structures. The iodine ion will be trapped in the coiled helical structures of starch and gives blue color. After digestion of starch by salivary amylase, no more helical structures available, and no color will be produced in addition of iodine. Therefore iodine can be taken as indicator to measure the activity of amylase, and sometimes, to find the inhibitor of amylase.

Nelson DDL, Cox MM (2005) Lehninger Principles of Biochemistry, 5th edition, W.H. Freeman and Co. Publisher, New York, USA, 248-252.

List of Experiments for Biochemistry Laboratory work

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                        List of Experiments for Laboratory work
Course Title: Laboratory work I: Biochemistry (Microbiology)
Credit: 1                                                                                                                                  

1. Preparation of normal, molar and percent solutions.
2. Preparation of different buffer solutions and measure their pH.
3. Determination of the isoelectric point of casein (of milk).
4. Titration of amino acids and find the physical constants.
5. Study the effects of temperature and pH on the enzymatic activity of salivary amylase.
6. Estimation of given protein concentration by Bradford, Lowry (Folin) protein assay and by Biuret test.
7. Qualitative detection of glucose in urine (the Trommer reaction).
8. Quantitative determination of glucose in blood by a glucose-oxidase method.
9. The determination of activity of the Krebs cycle dehydrogenases in the liver.
10. Determination of total cholesterol concentration in serum.
11. Determination of urea concentration in serum and urine.
12. Quantitative determination of phospholipids concentration in serum.
13. Determination of saponification value of the fat.
14. Separation of amino acids by chromatographic techniques and detection by nin-hydrin test. 

Identification of Beta-lactamase Inhibitor; a Strategy for Drug Development against Antibiotics Resistant Bacteria

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Introduction:                                                                             By Bishnu Marasini

       The most threatening to human health is due to bacterial infection and intensive research has been addressing to treat these maladies since the known history. The infectious bacteria are microscopic, unicellular prokaryotes and different than mammalian eukaryotic cells. The outer layer of the bacterial cell consists of cell wall which is not found in mammalian cell and can be taken as target for development of bactericidal agents. The finding of clear zone of inhibition of bacterial culture around the growth of Penicillium notatum in the experiment of Alexander Fleming in 1928 was the positive indication to get rid of such threat.

       Nowadays almost all bacterial infection can be cured using antibiotics. The world consumes tons of antibiotics per year and half of them are beta-lactam type e.g. penicillin, amoxicillin, cephalosporin, cephalexin, cefixime, ceftriaxome, monobactam, carbapenem, methicillin etc. Beta-lactam antibiotics have broad spectrum activity, economical friendly on production, good safety profile, have clinical efficacy. It is also target specific for prokaryotic cells, little side effects except some allergic reaction. So, it has gained wide popularity.

       One of the components of bacterial cell wall is peptidoglycan which is cross linked polymer of repeatedly units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM). The final step in cell wall biosynthesis is transamidation reaction catalyzed by the enzyme cell wall transamidase (CWT) also called as penicillin binding protein (PBP). This enzyme helps to cross link the polymer of NAG and NAM. The highly strained and reactive beta-lactam ring of the antibiotics reacts and binds irreversibly with the serine hydroxyl group of PBP (shown in figure 1) which inactivates the enzyme and ultimately death of bacteria (Frère et al, 1984; Tipper and Strominger, 1965).

Figure1: Binding of β-lactam Ring with Penicillin Binding Protein

       However, beta-lactam antibiotics are becoming ineffective against pathogenic bacteria. The most common reason is due to the production of beta-lactamase enzyme (EC 3.5.2.6) which catalyze the hydrolysis of the antibiotics i.e., formation of carboxyl group degrading beta-lactam ring (Shown in figure 2). Hydrolyzed antibiotics lose its activity or binding affinity towards the PBP hence no effect against bacteria. Hydrolysis of beta-lactam is rapid by beta-lactamase than binding of beta-lactam to PBP (Bush 1988). 

Figure 2: Degradation of β-lactam ring by β-lactamase enzyme

       The beta-lactamse (penicillinase) was reported just few years after the first antibiotic discovered (Abraham and Chain, 1940). Although penicillin is the oldest antibiotic and most of the organisms acquired resistant, it is first therapeutic choice in some diseases like syphilis. Beta-lactamse enzyme is an extra cellular enzyme in Gram-positive bacteria and found in periplasmic membrane in Gram-negative bacteria (Bowden and Georgiou, 1990; Dyke and Richmond, 1967). More than 200 types of beta-lactamse have been found (Bush et al, 1995). The difference among them is only the catalytic efficacy and turn over rate range from 0.004 to 1,200 molecules per second by 1 molecule of enzyme. Among them two types i.e. penicillinase and cephalosporinase type has a potent influence on the profile of the beta-lactam resistant antibiotics. Class A beta-lactamse has high affinity towards penicillin G but low affinity towards cephalosporin while class C beta-lactamse has opposite. Class B beta-lactamse hydrolyze the antibiotics by binding with the co-factor zinc (Zn) and class A, C and D hydrolyze by binding through serine residue of it to beta-lactam ring (Sawai et al, 1981). Beta-lactamase became widespread via the mechanism of plasmid exchange/insert among the pathogens (Sykes and Richmond, 1970). The rapid spread and evolution of these enzymes have seriously threatened the present antimicrobial arsenal.

       Two strategies have been developed to combat the problem of resistant. The first approach has been the synthesis/production of beta-lactamase resistant antibiotics e.g. penicillinase resistant beta-lactam antibiotics, nafcillin, oxacillin, ceftriaxone, cefoxitime, aztreonam, imipenem etc. But after few exposure to pathogens these antibiotics also become susceptible to extended spectrum beta-lactamase (ESBL) produced by multi-drug resistant (MDR) pathogens.

       The second approach is to use beta-lactamase inhibitors coupled with beta-lactam antibiotics. These enzyme inhibitors function to permanently inactivate the beta-lactamase in the periplasmic space so that the partner antibiotics can reach its target, penicillin binding protein (PBP). Broad spectrum beta-lactam antibiotics plus beta-lactamase inhibitors combination have been found good safety records and clinical efficacies (Munoz et al, 1996). Augmentin, the production of GlaxoSmithKline which is composed of amoxicillin and clavulanate in 2:1; Timentin (ticarcillin and clavulanate); Sultamicillin (ampicillin and sulbactam) are examples of beta-lactamase inhibitors in combination with beta-lactam used clinically. Clavulanate exhibited clinical efficacy than others and used as standard inhibitor. However, clavulanate was not found so effective against class C beta-lactamase (cephalorinase type) (Bush K, 1989). It also exhibited side effects on the long term of use like liver function destruction, gastrointestinal toxicity etc. (Ioannidis et al, 2002). Also it contains beta-lactam ring it and may be susceptible to beta-lactamase enzyme in upcoming days as broad spectrum beta-lactam antibiotics which were resistant to beta-lactamase, now become susceptible.