Michaelis–Menten kinetics

Name: Aaron Cummins ID Number: 16185315 Course: Industrial Biochemistry Department: CES Module: BC4904 Title: Determination of the KM, VMax for an enzymatic reaction: The reaction of Trypsin with Benzoyl-Arginine-P-Nitroanalide (BAPNA) Introduction: (Definitions) KM: The substrate concentration at 1/2 the maximum velocity. Relates to the affinity the substrate has for the enzyme VMAX: The maximum velocity or rate at which the enzyme catalyzed a reaction. Amount of substrate converted to

and methods 3 Data and calculations 5 Record of all raw data (results) 5 Calculations 5 Results and discussion 16 Interpretation 16 Discussion of significance of result 17 Conclusions 18 References 18 Introduction Aim: to determine the kinetic parameters of the enzyme Alkaline phosphatase General Background Alkaline Phosphatase is a group of enzymes found in certain tissues (including the bile duct, bones and the liver). Based on its specific location each enzyme will present with a

para-Nitrophenylphosphate (pPNP) is an example of a chromogenic substrate of this enzyme, because the product of hydrolysis of pPNP is the yellow para-nitrophenol (pNP) compound along with inorganic phosphate (McComb, Bowers and Posen, 1979). The Michaelis–Menten equation for enzyme kinetics (Murray, 2002) predicts an initially high rate of enzyme activity as the concentration of the substrate is relatively high, followed by a gradual reduction in enzyme activity as the substrate is consumed. The enzyme alkaline phosphatase

Figure 5 - Michaelis Menten Graph of Ethanol (pH 9.0) Representing the Velocity versus Substrate Concentration. Different concentrations of ethanol were used, and the absorbance reading taken through enzyme kinetics. The velocity was calculated through the conversion of the slope of the data points to abs/min. K_m was found to be 5.76×〖10〗^(-3) M, V_max 1.71×〖10〗^(-3) M/min, K_cat 2.85×〖10〗^(-2) 〖min〗^(-1), and K_cat/K_m 4.94 〖min〗^(-1) M^(-1). Figure 5 - Michaelis Menten Graph of Ethanol (pH

The concentrations of L-arginine and L-asparagine were measured in the range 0.110 mM. Data was collected from the fluorescence intensity of the Arg-and Asn-sensing membranes at the two emission wavelengths (λem = 565 and 625 nm) at an excitation wavelength of 460 nm (λex = 460 nm). The fluorescence spectra for detecting L-Arg and L-Asn were measured using a multifunctional fluorescence microtiter plate reader (Saphire2, Tecan GmbH, Wien, Austria). The Arg-sensing membranes immobilized with various

and volume of hydrogen peroxide solution are kept constant. It was expected that if the concentration of hydrogen peroxide increased, then the production rate of oxygen gas would also increase until the enzyme became saturated, following the Michaelis Menten relationship. For this to apply, the volume and temperature of 50% catalase and volume of hydrogen peroxide solution were kept constant. Six different concentrations were used

Biology, 1971 University Blvd, Lynchburg, VA 24502. Materials and Methods Dilution: Alkaline Phosphatase The objective for this laboratory is to determine the velocity of alkaline phosphatase and make a graph of rate vs substrate concentration (Michaelis Menten plot). After the wavelength was determined the para-Nitrophenyl phosphate (PNPP, Sigma Aldrich) was obtained to get the absorption spectrum. To obtain a reaction of .5 A/min, a 0.5 of substrate solution was used and 0.015-0.07 ml of enzyme solution

Enzymes are complex molecular machines that deal with a broad array of chemical mechanisms. According to Michaelis-Menten kinetics, enzyme-substrate reactions are composed of two reactions. The first reaction occurs when the substrate forms a complex with the enzyme. The second reaction utilizes the complex previously made to decompose into enzyme and product. When

Therefore, to determine those various constants, kinetic enzymatic activity must be measured. The absorbance will be measured using a a Genesys 10S UV-VIS spectrophotometer which uses VISIONlite software. Fortunately the substrate specificity of the enzyme is such that a variety of different substrates

Enzymes are large biological molecules responsible for the thousands of chemical interconversions that sustain life. They are highly selective catalysts, greatly accelerating both the rate and specificity of metabolic reactions, from the digestion of food to the synthesis of DNA. Most enzymes are proteins, although some catalytic RNA molecules have been identified. Enzymes adopt a specific three-dimensional structure, and may employ organic (e.g. biotin) and inorganic (e.g. magnesium ion) cofactors