Abstract
Enzymes are specific biocatalysts which enhance the rate of biological reactions in mild conditions of temperature, pressure and a particular pH and thus have an important role in almost every cellular process. They play an indispensable role in metabolism, signal transduction cascade, and gene expression which makes them an important target for drug development, bioprocess industry, food industry, and eco-friendly green chemistries. They are emerging as potential drug targets. Many diseases are been explored where enzyme inhibition can have therapeutic effects. The study and analysis of enzyme have been challenging due to its mathematical aspects. However, due to their important functions, it becomes imperative to have sufficient and thorough knowledge of biochemistry of an enzyme and deep understanding of kinetics of enzyme action. In the present chapter, we will explore very simple and established basic concept of enzyme kinetics, estimation of initial reaction velocities with different substrate concentration, measurement and utility of KM and Vmax, catalytic power of the enzyme along with its turnover number. The readers will learn about changes which occur in their graph with different enzyme inhibitors in their reaction. This chapter would also be helpful for researchers and students to understand positive and negative feedback loop. We have provided example of serum alkaline phosphatase (ALP) where one can learn to establish different parameters with increase in absorbance as substrate concentration increases.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Jeremy MB, John LT, Lubert S (2002) Biochemistry, 5th edn. W H Freeman, New York; ISBN-10: 0-7167-3051-0
Voet D, Voet JG, Pratt CW (2013) Fundamentals of biochemistry: life at the molecular level, 4th edn. Wiley, Hoboken, NJ; ISBN 13: 9780470570951
Michaelis L, Menten M (1913) Die kinetik der invertinwirkung. Biochem Z 49:333–369
Sakharov IY, Makarova IE, Ermolin GA (1988) Purification and characterization of intestinal alkaline phosphatase from harp seal. Comp Biochem Physiol B 90:709–714
Lehninger AL, Nelson DL, Cox MM (2000) Lehninger principles of biochemistry. Worth Publishers, New York
Kapojos J, Poelstra K, Borghuis T, Van den Berg A, Baelde H, Klok P, Bakker W (2003) Induction of glomerular alkaline phosphatase after challenge with lipopolysaccharide. Int J Exp Pathol 84:135–144
Poelstra K, Bakker W, Klok P, Hardonk M, Meijer D (1997) A physiologic function for alkaline phosphatase: endotoxin detoxification. Lab Investig 76:319–328
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56(3):658–666. https://doi.org/10.1021/ja01318a036
Eadie GS (1942) The inhibition of cholinesterase by Physostigmine and Prostigmine. J Biol Chem 146:85–93
Hofstee BHJ (1959) Non-inverted versus inverted plots in enzyme kinetics. Nature 184(4695):1296–1298. https://doi.org/10.1038/1841296b0
http://docs.abcam.com/pdf/protocols/Troubleshooing-guide-for-enzymatic-assay-kits.pdf
Bisswanger H, (2014) Enzyme assays. Perspectives in Science 1(1–6):41–55
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gupta, V., Gupta, A., Shukla, L.I., Jha, A.K., Prakash, J., Tripathy, B.C. (2020). Determination of Michaelis–Menten Enzyme Kinetics Parameters of Alkaline Phosphatase in Clinical Samples. In: Gupta, N., Gupta, V. (eds) Experimental Protocols in Biotechnology. Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0607-0_14
Download citation
DOI: https://doi.org/10.1007/978-1-0716-0607-0_14
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-0606-3
Online ISBN: 978-1-0716-0607-0
eBook Packages: Springer Protocols