DOCSLIB.ORG

Enzyme Kinetics

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Enzyme Kinetics Enzyme Kinetics… Virtually All Reactions in Cells Are Mediated by Enzymes • Enzymes catalyze thermodynamically favorable reactions, causing them to proceed at extraordinarily rapid rates • Enzymes provide cells with the ability to exert kinetic control over thermodynamic potentiality • Living systems use enzymes to accelerate and control the rates of vitally important biochemical reactions • Enzymes are the agents of metabolic function Enzymatic catalysis Role of enzymes • serve the same role as any other catalyst in chemistry • act with a higher specificity • acid and base catalyst possible due to proximal locations of amino acids • alter the structure location of quantity of the enzyme and you have regulated the formation of product - try that in organic chemistry ! • Don't lose sight of what enzymes do. catalyze reactions – nothing more reactants -> products. What do Enzyme ACTUALLY do? Naming Enzymes •Enzyme commission for each enzyme based on the type of reactions. Kind of like IUPAC •Yeah right - the mother of invention – he/she who finds/discovers/purifies/clones the enzyme, names if. Trivial names rule… • First recognition of an enzyme and naming – 1833: alcohol precipitate of malt extract converted starch into sugar – called diatase (first use of suffix –ase) • Proteases traditionaly are the only enzymes which don’t end in ‘ase’ and use ‘-in’ • Enzyme is coined after the Greek for “in yeast” • 1050s started need to use a standardized naming: Otto Hofmann-Ostenhof and Dixon & Webb – common, non-systematic names still rule. Enzyme Nomenclature Provides a Systematic Way of Naming Metabolic Reactions O T H L I L Enzyme reaction types: Enzymes are classified into six major classes based on the reaction they catalyze. 1) Oxidoreductases catalyze oxidation-reduction reactions. Most of these enzymes are known as dehydrogenases, but some are called oxidases, peroxidases, oxygenases or reductases. 2) Transferases catalyze group-transfer reactions (functional groups). Many require the presence of coenzymes. A portion of the substrate molecule usually binds covalently to these enzymes or their coenzymes. This group includes the kinases! 3) Hydrolases catalyze hydrolysis. They are a special class of transferases, with water serving as the acceptor of the group transferred 4) Lyases catalyze nonhydrolytic and nonoxidative elimination reactions, or lysis, of a substrate, generating a double bond. In other words these reactions catalyze group elimination to form double bonds. A lyase that catalyzes addition reaction in cells is often termed a synthase. 5) Isomerases catalyze the isomerase reactions. Because these reactions have only one substrate and one product, they are among the simplest enzymatic reactions. 6) Ligases catalyze ligation or joining, of two substrates. These reactions require the input of chemical potential energy of a nucleoside triphosphate such as ATP. i.e. bond formation coupled to ATP hydrolysis. Ligases are usually referred to as synthetases. Often involved in joining carbon atoms to N, S or O atoms. 1 Enzyme specificity - One of the most powerful actions of enzymes. – Group complementation - the ability to recognize specific regions of the substrate to align reactants with catalytic site. – Based on non-covalent molecular interactions. – Lock and key vs. induced fit - both occur. Induced fit takes place when binding of one part of the substrate to the enzyme alters the conformation of the enzyme to make a true "fit" – Binding does not mean activity Enzyme specificity An enzyme can bind and react stereo-specifically with chiral compounds This can happen due to a three point attachment – Binding can then only occur in one way and therefore the products are not a mixture. General Information on Enzymology Enzyme nomenclature • Active site • Substrate vs. reactant • Prosthetic groups, coenzyme and cofactors • Activity vs. reaction • Suffix -ase Another important reminder - not all activity is on or off. Many times the enzyme has a low or constitutive activity that can be increased many times. It is a common mistake to think of enzymes being on or off. Job of Enzyme Catalysts • Specifically bind substrate(s) (reactants), decrease transition state energy to increase the rate (NOT thermodynamics) of a favorable reaction: • High specificity • Low side reactions • Ability to be regulated: Active site – amino acids and cofactors binding and “doing” the reaction, are regulated by the rest of the protein How do enzymes work? • By reducing the energy of activation • This happens because the transition state is stabilized by a number of mechanisms involving the enzyme/protein • Without enzymes reactions occur by collisions between reactants or addition of various organic catalysts • The energy barrier between the reactants and product, is the free energy of activation. • The free energy (DG) of the reaction is what determines if a reaction is spontaneous. Reactions are often multistep Two intermediate transition states. • Two step reactions will have a slow and fast step – based on activation energy • The higher activation step is the rate-limiting step of the reaction Enzyme Kinetics • Kinetics is the branch of science concerned with the rates of reactions • Enzyme kinetics seeks to determine the maximum reaction velocity that enzymes can attain and the binding affinities for substrates and inhibitors • Analysis of enzyme rates yields insights into enzyme mechanisms and metabolic pathways • This information can be exploited to control and manipulate the course of metabolic events 2 Rates of a reaction… Molecularity – the number of molecules that must collide/interact for a reaction to occur. • For enzymes, molecules that are involved at the active site for a step of the reaction. • Unlike non-enzyme catalyzed reactions, the rate is too complex but often initially described as unimolecular or bimolecular • Remember, order – refers to rate equation, while molecularity defines the mechanism Gen Chem – Rates of Reaction… Quick Review – MOST enzymes catalyze reactions in a first or second order reaction. - Less about collisions of freely moving molecules than binding and reacting on a surface of a catalyst (enzyme active site): For single/uni-molecularity reactions – 1st order A->P The rate is proportional to the concentration of substrate Enzyme Kinetics Kinetic defn.: Of or relating to or produced by motion. Kinetics defn.: the branch of chemistry or biochemistry concerned with measuring and studying the rates of reactions. Enzyme kinetics are defined differently than basic chemistry kinetics because of the large catalyst… Measuring Rates of Enzyme Catalyzed Reactions Louis Michaelis and Maud Menten's theory • assumes the formation of an enzyme-substrate complex • assumes that the ES complex is in rapid equilibrium with free enzyme • assumes that the breakdown of ES to form products is slower than 1) formation of ES and 2) breakdown of ES to re-form E and S Enzyme Kinetics An enzyme-catalyzed reaction of substrate S to product P, can be written Actually, the enzyme and substrate must combine and E recycled after the reaction is finished, just like any catalyst. Because the enzyme actually binds the substrate the reaction can be written as: The simplest reaction is a single substrate going to a single product. Rate or velocity of the reaction depends on the formation of the ES •The P -> ES is ignored •The equilibrium constant Keq is based on the idea that the reaction is limited to the formation of the ES complex and that only K1 and K-1 are involved because the thermodynamics of the reversal of K2 cause it to be minimal How fast an enzyme catalyzes a reaction is it's rate. The rate of the reaction is in the number of moles of product produced per second [ES] Remains Constant Through Much of the Enzyme Reaction Time Course in Michaelis-Menten Kinetics 3 Time course for a typical enzyme-catalyzed reaction obeying the Michaelis-Menten, Briggs-Haldane models for enzyme kinetics. • The early state of the time course is shown in greater magnification in the bottom graph. Enzyme-Catalyzed Reactions • The relationship between the concentration of a substrate and the rate of an enzymatic reaction is described by looking at the concentration of S and v • At low concentrations of the enzyme substrate, the rate is proportional to S, as in a first-order reaction • At higher concentrations of substrate, the enzyme reaction approaches zero-order kinetics • This behavior is a saturation effect Psuedo First Order Kinetics For the most part enzyme reactions are treated as if there is only one substrate and one product. If there are two substrates, one of them is held at a high concentration (0 order) and the other substrate is studied at a lower concentration • so that for that substrate, it is a first order reaction. This leads us to the M and M equation. •Mathematical model of the representation of the M&M eq. - For the reaction: 1) The Michaelis constant Km is: Think of what this means in terms of the equilibrium. Large vs. a small Km 2) When investigating the initial rate (Vo) the Michaelis-Menten equation is: Graphical representation is a hyperbola. Think of the difference between O2 binding of myoglobin and hemoglobin. •When [S] << Km, the velocity is dependent on [S] •When [S] >> Km, the initial velocity is independent of [S] •When [S] = Km, then Vo = 1/2 V max Understanding Vmax The theoretical maximal velocity Vmax is a constant Vmax is the theoretical maximal rate of the reaction - but it is NEVER achieved in reality To reach Vmax would require that ALL enzyme molecules are tightly bound with substrate Vmax is asymptotically approached as substrate is increased Understanding KmThe "kinetic activator constant" Km is a constant Km is a constant derived from rate constants Km is, under true Michaelis-Menten conditions, an estimate of the dissociation constant of E from S Small Km means tight binding; high Km means weak binding Conditions for Michaelis -Menten Two assumptions must be met for the Michaelis-Menten equation 1) Equilibrium -the association and dissociation of the substrate and enzyme 4 is assumed to be a rapid equilibrium and Ks is the enzyme:substrate dissociation constant.
Load more

Recommended publications
  • Chapter 4. Enzyme Kinetics
    Chapter 4. Enzyme Kinetics Enzyme Engineering 4.1 Why is enzyme kinetics important? 1. It provides valuable information for enzyme mechanism 2. It gives an insight into the role of an enzyme under physiological conditions 3. It can help show how the enzyme activity is controlled and regulated 1 4.2 How to obtain enzyme kinetics? Measure the formation of product or disappearance of substrate Mg2+ D-Glucose + ATP D-Glucose-6-phosphate + ADP 1. Discontinuous method Add the enzyme, then stop the reaction at different time by adding acid to the sample Measure the concentration of substrate or product using HPLC or other chromatography 4.2 How to obtain enzyme kinetics? 2. Continuous method Mg2+ D-Glucose + ATP D-Glucose-6-phosphate + ADP NADP+ NADPH D-Glucono-δ-lactone 6-phosphate + ADP NADPH absorb at 340nm, while NADP+ does not If a coupled reaction is used, the second reaction should not be rate-limiting (sufficient amount of the enzyme must be provided) Continuous method is not always available 2 4.2 How to obtain enzyme kinetics? Radioactive or fluorescent-labeling is useful Precautions The substrate, buffers, etc. must be extremely pure Enzyme should be pure Enzyme should be stable as the assay is proceeded * Temp, pH must be maintained carefully Once steady state is achieved, reaction rate must be constant and proportional to the enzyme added Initial rate of reaction should be measured to avoid possible complexity 4.3 How to analyze kinetic data? 4.3.1 One substrate reactions k 1 k2 E + S ES E + P k-1 Equilibrium assumption (1913) Second reaction is slower than first reverse reaction (k-1 >> k2) Vmax[S] V0 = Michaelis-Menten eqn K s +[S] [E][S] Vmax = k2[E]0 K = s [ES] 3 4.3 How to analyze kinetic data? Steady state assumption (1925) [ES] is constant Vmax[S] V0 = Km +[S] Vmax = k2[E]0 k2 + k−1 Km = k1 4.3.1 One substrate reactions Lineweaver-Burk equation 1 K 1 1 = m + v [S] Vmax Vmax Eddie-Hofstee equation v V v = max − [S] Km Km 4 4.3.1 One substrate reactions Significance of the result 1.
    [Show full text]
  • Catalase Kinetics
    Experiment #4: Catalase Kinetics MASSACHUSETTS INSTITUTE OF TECHNOLOGY Department of Chemistry 5.310 Laboratory Chemistry 1 EXPERIMENT #4 A Study of the Kinetics of the Enzyme Catalase and its Reaction 2 3 With H2O2. A Further Study on Protein Assay Quantitation of Catalase I. OVERVIEW OF THE EXPERIMENT In this experiment, the student will investigate the enzyme activity of catalase by studying the decomposition of H2O2 to form water and oxygen. Using an oxygen based pressure sensor the student measures the amount of oxygen produced and then calculates the rate of the enzyme catalyzed reaction under various conditions. The student also completes a Protein Assay on an unknown sample of catalase using the Coomassie® Plus Protein Assay from Pierce to determine the protein concentration of the sample. The correlation between the actual and calculated concentration gives an indication of the experimental skills used in carrying out the experiment. II. OBJECTIVES This is an integrated experiment that includes topics from physical chemistry and biochemistry. It is designed to introduce students to the basics of: • Studying the effects of reaction environment, including temperature, and varying substrate concentration on the rate of an enzyme-catalyzed reaction. • Combining physical chemistry and biochemistry, principles and theory, with the goal of determining various biochemical and biophysical constants for the enzyme catalase. • How to acquire experimental kinetic data for an enzyme catalyzed reaction. • Learning how to perform data manipulation in order to extract out information such as rate constants from experimental kinetic data. • Correct handling of UV-VIS Spectroscopy 1 This experiment was synthesized by John J.
    [Show full text]
  • Estimation of the Overall Kinetic Parameters of Enzyme Inactivation Using an Isoconversional Method D
    Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan To cite this version: D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan. Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method. Biophysical Chemistry, Elsevier, 2008, 138 (1-2), pp.50. 10.1016/j.bpc.2008.09.003. hal-00501718 HAL Id: hal-00501718 https://hal.archives-ouvertes.fr/hal-00501718 Submitted on 12 Jul 2010 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. ÔØ ÅÒÙ×Ö ÔØ Estimation of the overall kinetic parameters of enzyme inactivation using an isoconversional method D. Oancea, Alexandrina Stuparu, Madalina Nita, Mihaela Puiu, Adina Raducan PII: S0301-4622(08)00176-2 DOI: doi: 10.1016/j.bpc.2008.09.003 Reference: BIOCHE 5155 To appear in: Biophysical Chemistry Received date: 17 August 2008 Revised date: 2 September 2008 Accepted date: 3 September 2008 Please cite this article as: D. Oancea, Alexandrina Stuparu, Madalina Nita, Mi- haela Puiu, Adina Raducan, Estimation of the overall kinetic parameters of en- zyme inactivation using an isoconversional method, Biophysical Chemistry (2008), doi: 10.1016/j.bpc.2008.09.003 This is a PDF file of an unedited manuscript that has been accepted for publication.
    [Show full text]
  • Determination of Enzyme Kinetics in the Eppendorf Biospectrometer® Kinetic Using a Hexokinase and Glucose-6- Phosphate-Dehydrogenase from Saccharomyces Cerevisiae
    USERGUIDE No. 39 I Detection I September 2014 Determination of enzyme kinetics in the Eppendorf BioSpectrometer® kinetic using a hexokinase and glucose-6- phosphate-dehydrogenase from Saccharomyces cerevisiae Martin Armbrecht, Eppendorf AG, Hamburg, Germany Abstract This Userguide will demonstrate measurement of enzyme activity using the Eppendorf BioSpectrometer. In order to optimize the measurement process, preliminary measurements were performed using the method “single continuous”. The actual activity measurements were then performed based on these results. Enzyme activities were determined via linear regression. For the measurements, a coupled reaction of a hexokinase and a glucose-6-phosphate-dehydrogenase from the baker’s yeast ‚ was used. Since the Eppendorf BioSpectrometer kinetic is equipped with a temperature controlled cuvette chamber, temperature dependence of this reaction could also be demonstrated. The highest activity was detected at 37 °C. Introduction Metabolic significance of the hexokinase reaction Activation of glucose via the hexokinase reaction Glycolysis Glucose degradation thus sustains respiration of all living beings. One central area within the metabolism of nearly all living beings is Prior to degradation, glucose needs to be rendered susceptible for the enzymatic degradation of glucose in the cytosol, or cytoplasma, degradation, i.e., activated. This process involves phosphorylation by respectively, in their cells. This process is called glycolysis. Glucose a hexokinase; with the expenditure of ATP, glucose-6-phosphate is is converted to 2 molecules of pyruvate in a stepwise fashion. One produced. Thus, the latter is the actual initial substrate of glycolysis molecule of glucose yields 2 ATP and 2 redox equivalents in the form (fig.1). In addition, glucose-6-phosphate is the initial substrate of a further of NADPH2.
    [Show full text]
  • Chemical Kinetics HW1 (Kahn, 2010)
    Chemical Kinetics HW1 (Kahn, 2010) Question 1. (6 pts) A reaction with stoichiometry A = P + 2Q was studied by monitoring the concentration of the reactant A as a function of time for eighteen minutes. The concentration determination method had a maximum error of 6 M. The following concentration profile was observed: Time (min) Conc (mM) 1 0.9850 2 0.8571 3 0.7482 4 0.6549 5 0.5885 6 0.5183 7 0.4667 8 0.4281 9 0.3864 10 0.3557 11 0.3259 12 0.3037 13 0.2706 14 0.2486 15 0.2355 16 0.2188 17 0.2111 18 0.1930 Determine the reaction order and calculate the rate constant for decomposition of A. What can be said about the mechanism or molecularity of this reaction? Question 2. (4 pts) Solve problem 2 on pg 31 in your textbook (House) using both linear and non-linear regression. Provide standard errors for the rate constant and half-life based on linear and non-linear fits. Below is the data set for your convenience: dataA = {{0, 0.5}, {10, 0.443}, {20,0.395}, {30,0.348}, {40,0.310}, {50,0.274}, {60,0.24}, {70,0.212}, {80,0.190}, {90,0.171}, {100,0.164}} Question 3. (3 pts) Solve problem 3 on pg 32 in your textbook (House). Question 4. (7 pts) The authors of the paper “Microsecond Folding of the Cold Shock Protein Measured by a Pressure-Jump Technique” suggest that the activated state of folding of CspB follows Hammond- type behavior.
    [Show full text]
  • Effect of Enzyme/Substrate Ratio on the Antioxidant Properties of Hydrolysed African Yam Bean
    African Journal of Biotechnology Vol. 11(50), pp. 11086-11091, 21 June, 2012 Available online at http://www.academicjournals.org/AJB DOI: 10.5897/AJB11.2271 ISSN 1684–5315 ©2012 Academic Journals Full Length Research Paper Effect of enzyme/substrate ratio on the antioxidant properties of hydrolysed African yam bean Fasasi Olufunmilayo*, Oyebode Esther and Fagbamila Oluwatoyin Department of Food Science and Technology, P. M. B. 704, Federal University of Technology, Akure, Nigeria. Accepted 8 June, 2012 The use of natural antioxidant as compared with synthetic antioxidant in food processing is a growing trend as consumers prefer natural to synthetic antioxidant mainly on emotional ground. This study investigates the antioxidant activity of hydrolysed African yam bean (Sphenostylis sternocarpa) which is regarded as one of the neglected underutilized species (NUS) of crop in Africa and Nigeria especially to improve food security and boost the economic importance of the crop. The antioxidant properties of African yam bean hydrolysates (AYH) produced at different enzyme to substrate (E/S) ratios of 1: 100 and 3: 100 (W/V) using pepsin (pH 2.0, 37°C) were studied. 2, 2-Diphenyl-1-picryl-hydrazyl (DPPH) radical scavenging activity of the hydrolysates was significantly influenced by the E\S ratio as DPPH radical scavenging activity ranged from 56.1 to 75.8% in AYH (1: 100) and 33.3 to 58.8% in AYH (3:100) with 1000 µg/ml having the highest DPPH radical scavenging activity. AYH (1:100) and AYH (3:100) had higher reducing activities of 0.42 and 0.23, respectively at a concentration of 1000 µg/ml.
    [Show full text]
  • Regulation of Energy Substrate Metabolism in Endurance Exercise
    International Journal of Environmental Research and Public Health Review Regulation of Energy Substrate Metabolism in Endurance Exercise Abdullah F. Alghannam 1,* , Mazen M. Ghaith 2 and Maha H. Alhussain 3 1 Lifestyle and Health Research Center, Health Sciences Research Center, Princess Nourah bInt. Abdulrahman University, Riyadh 84428, Saudi Arabia 2 Faculty of Applied Medical Sciences, Laboratory Medicine Department, Umm Al-Qura University, Al Abdeyah, Makkah 7607, Saudi Arabia; [email protected] 3 Department of Food Science and Nutrition, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia; [email protected] * Correspondence: [email protected] Abstract: The human body requires energy to function. Adenosine triphosphate (ATP) is the cellular currency for energy-requiring processes including mechanical work (i.e., exercise). ATP used by the cells is ultimately derived from the catabolism of energy substrate molecules—carbohydrates, fat, and protein. In prolonged moderate to high-intensity exercise, there is a delicate interplay between carbohydrate and fat metabolism, and this bioenergetic process is tightly regulated by numerous physiological, nutritional, and environmental factors such as exercise intensity and du- ration, body mass and feeding state. Carbohydrate metabolism is of critical importance during prolonged endurance-type exercise, reflecting the physiological need to regulate glucose homeostasis, assuring optimal glycogen storage, proper muscle fuelling, and delaying the onset of fatigue. Fat metabolism represents a sustainable source of energy to meet energy demands and preserve the ‘limited’ carbohydrate stores. Coordinated neural, hormonal and circulatory events occur during prolonged endurance-type exercise, facilitating the delivery of fatty acids from adipose tissue to the Citation: Alghannam, A.F.; Ghaith, working muscle for oxidation.
    [Show full text]
  • Detection of Errors of Interpretation in Experiments in Enzyme Kinetics
    METHODS 24, 181±190 (2001) doi:10.1006/meth.2001.1179, available online at http://www.idealibrary.com on Detection of Errors of Interpretation in Experiments in Enzyme Kinetics Athel Cornish-Bowden1 Institut FeÂdeÂratif ªBiologie Structurale et Microbiologie,º BioeÂnergeÂtique et IngeÂnierie des ProteÂines, Centre National de la Recherche Scientifique, 31 chemin Joseph-Aiguier, B.P. 71, 13402 Marseille Cedex 20, France points necessary for modern users are to be aware (at Although modern statistical computing will often be the method least qualitatively) of the assumptions that underlie of choice for analyzing kinetic data, graphic methods provide an important supplement that ought not to be neglected. Residual the statistical analysis and to be aware also that they plots, or plots of differences between observed and calculated are not always true (2). By contrast we shall be con- values against variables not expected to be correlated with these cerned with methods that require no detailed mathe- differences, permit a rapid judgment of whether data have been matics and can readily be applied to published graphs correctly interpreted and analyzed. The rapid increase in the fre- (or even to graphs displayed on a screen during a lec- quency with which artificially modified or mutated enzymes are ture) without access to the numerical coordinates of studied is making it less and less safe to assume that enzymes the observations. More generally, this article argues for are stable under assay conditions, and there is thus an increased more reliance on common sense and graphic analysis need for methods to check for enzyme stability, and a method and less on automatic analysis performed by computer: for doing this is briefly described.
    [Show full text]
  • NEW TANDEM REACTIONS INVOLVING NUCLEOPHILIC AROMATIC SUBSTITUTION by JAMES ERVIN SCHAMMERHORN III Associates of Science Murray
    NEW TANDEM REACTIONS INVOLVING NUCLEOPHILIC AROMATIC SUBSTITUTION By JAMES ERVIN SCHAMMERHORN III Associates of Science Murray State College Tishomingo, Oklahoma 2003 Bachelor of Science in Chemistry Oklahoma State University Stillwater, Oklahoma 2006 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY May, 2011 1 NEW TANDEM REACTIONS INVOLVING NUCLEOPHILIC ARMOMATIC SUBSTITUTION Thesis Approved: Dr. Richard Bunce Thesis Adviser Dr. K. Darrell Berlin Dr. Ziad El-Rassi Dr. Nicholas Materer Dr. Andrew Mort Dr. Mark E. Payton Dean of the Graduate College ii ACKNOWLEDGMENTS I would like to express my sincere gratitude to Dr. R. A. Bunce, my research advisor, my graduate committee chairman and my friend. I have learned many valuable lessons working alongside him in his research laboratory. His ability to teach both organic theory and laboratory techniques have been instrumental in my research at Oklahoma State University. I am immensely thankful for his support, guidance and patience to me during the course of this research. His unique sense of humor always makes me laugh and makes it a joy to come to lab. I would also like to thank my committee members, Drs. K. D. Berlin, Z. El Rassi, N. F. Materer, and A. J. Mort their acceptance to be on my committee. Their insights into research and graduate life have been invaluable during my graduate career. I am especially grateful to Dr. Berlin for sharing his wealth of organic chemistry knowledge with me during the course of my research. Also, to Dr.
    [Show full text]
  • Chromatographic Resolution and Kinetic Characterization Of
    Proc. NatL Acad. Sci. USA Vol. 80, pp. 8589, January 1983 Biochemistry Chromatographic resolution and kinetic characterization of glucokinase from islets of Langerhans (hexoldnase/islet glucose metabolism /N-acetylglucosamine kdnase/Cibacron blue) MARTIN D. MEGLASSON, PAMELA TRUEHEART BURCH, DONNA K. BERNER, HABIBA NAJAFI, ALAN P. VOGIN, AND FRANZ M. MATSCHINSKY* Diabetes Research Center and Department of Biochemistry and Biophysics, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Communicated by Robert E. Forster, October 4, 1982' ABSTRACT Glucokinase (ATP:D-glucose 6-phosphotransfer- ofislets equals serum glucose and islet glucose 6-phosphate in- ase, EC 2.7.1.2) from rat islets of Langerhans was partially pu- creases in proportion to extracellular glucose (1, 13). Ninety rified by chromatography on DEAE-Cibacron blue F3GA agar- percent ofislet glucose utilization occurs with an apparent glu- ose. The enzyme eluted in two separate peaks. Sigmoidal rate cose affinity constant 11.1 mM (6). Also, mannoheptulose, an dependence was found with respect to glucose (Hill coefficient inhibitor of glucokinase (8), profoundly inhibits islet glucose = 1.5) for both enzyme fractions. K. values for glucose were 5.7 metabolism. (14). mM for the major fraction and 4.5 mM for the minor fraction. The chromatographic separation of glucokinase from other Neither fraction phosphorylated GlcNAc. A' GlcNAc kinase is the most direct (ATP. 2-acetamido-2-deoxy-D-glucose' 6-phosphotransferase; EC glucose phosphorylating enzymes approach 2.7.1.59)-enriched fraction; prepared by affinity chromatography to establish its presence in pancreatic islets. It is the purpose on Sepharose-N46-aminohexanoyl)-GlcNAc, had a K.
    [Show full text]
  • Spring 2013 Lecture 13-14
    CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna INTRODUCTION TO ENZYMES • Enzymes are usually proteins (some RNA) • In general, names end with suffix “ase” • Enzymes are catalysts – increase the rate of a reaction – not consumed by the reaction – act repeatedly to increase the rate of reactions – Enzymes are often very “specific” – promote only 1 particular reaction – Reactants also called “substrates” of enzyme catalyst rate enhancement non-enzymatic (Pd) 102-104 fold enzymatic up to 1020 fold • How much is 1020 fold? catalyst time for reaction yes 1 second no 3 x 1012 years • 3 x 1012 years is 500 times the age of the earth! Carbonic Anhydrase Tissues ! + CO2 +H2O HCO3− +H "Lungs and Kidney 107 rate enhancement Facilitates the transfer of carbon dioxide from tissues to blood and from blood to alveolar air Many enzyme names end in –ase 89 CHM333 LECTURE 13 – 14: 2/13 – 15/13 SPRING 2013 Professor Christine Hrycyna Why Enzymes? • Accelerate and control the rates of vitally important biochemical reactions • Greater reaction specificity • Milder reaction conditions • Capacity for regulation • Enzymes are the agents of metabolic function. • Metabolites have many potential pathways • Enzymes make the desired one most favorable • Enzymes are necessary for life to exist – otherwise reactions would occur too slowly for a metabolizing organis • Enzymes DO NOT change the equilibrium constant of a reaction (accelerates the rates of the forward and reverse reactions equally) • Enzymes DO NOT alter the standard free energy change, (ΔG°) of a reaction 1. ΔG° = amount of energy consumed or liberated in the reaction 2.
    [Show full text]
  • Reactions of Aromatic Compounds Just Like an Alkene, Benzene Has Clouds of  Electrons Above and Below Its Sigma Bond Framework
    Reactions of Aromatic Compounds Just like an alkene, benzene has clouds of electrons above and below its sigma bond framework. Although the electrons are in a stable aromatic system, they are still available for reaction with strong electrophiles. This generates a carbocation which is resonance stabilized (but not aromatic). This cation is called a sigma complex because the electrophile is joined to the benzene ring through a new sigma bond. The sigma complex (also called an arenium ion) is not aromatic since it contains an sp3 carbon (which disrupts the required loop of p orbitals). Ch17 Reactions of Aromatic Compounds (landscape).docx Page1 The loss of aromaticity required to form the sigma complex explains the highly endothermic nature of the first step. (That is why we require strong electrophiles for reaction). The sigma complex wishes to regain its aromaticity, and it may do so by either a reversal of the first step (i.e. regenerate the starting material) or by loss of the proton on the sp3 carbon (leading to a substitution product). When a reaction proceeds this way, it is electrophilic aromatic substitution. There are a wide variety of electrophiles that can be introduced into a benzene ring in this way, and so electrophilic aromatic substitution is a very important method for the synthesis of substituted aromatic compounds. Ch17 Reactions of Aromatic Compounds (landscape).docx Page2 Bromination of Benzene Bromination follows the same general mechanism for the electrophilic aromatic substitution (EAS). Bromine itself is not electrophilic enough to react with benzene. But the addition of a strong Lewis acid (electron pair acceptor), such as FeBr3, catalyses the reaction, and leads to the substitution product.
    [Show full text]