Kinetically Perfect Enzymes

A diffusion-limited enzyme catalyses a reaction so efficiently that the rate limiting step is that of substrate diffusion into the active site, or product diffusion out. This is also known as kinetic perfection or catalytic perfection. Since the rate of catalysis of such enzymes is set by the diffusion-controlled reaction, it therefore represents an intrinsic, physical constraint on evolution (a maximum peak height in the fitness landscape). Diffusion limited perfect enzymes are very rare. Most enzymes catalyse their reactions to a rate that is 1,000-10,000 times slower than this limit. This is due to both the chemical limitations of difficult reactions, and the evolutionary limitations that such high reaction rates do not confer any extra fitness.

1. Kinetically perfect enzymes Presented by, SHRYLI K S V th Semester YMB17118 Molecular Biology, Yuvaraja's College (Autonomous), Mysuru Guided by, Dr. Ragavendra Hegade Katte Guest Faculty Dept. Molecular Biology, Yuvaraja's College (Autonomous), Mysuru 24 t May, 2020 MINOR SEMINAR Enzymology

2. Contents Introduction Michaelis - Menten equation. K inetically perfect enzymes. A dvantages of kinetically perfect enzymes with respect to biological systems. Triose phosphate isomerase. Acetylcholinesterase. Superoxide Dismutase. Conclusion References Acknowledgement 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 2

3. Introduction Chemical Kinetics- Branch of physical chemistry that is concerned with understanding the rate of chemical reactions. Enzymes are the biological catalyst that play a critical role in accelerating reactions anywhere from 10 3 to 10 17 times faster than the normal rate of the reaction. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 3

4. Michaelis - Menten Enzyme Kinetics Fig 01: An enzyme catalyzes the reaction of two substrates and to form one product. This can be described with the following multistep mechanism. 1 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 4

5. Where k 1 , k –1 , k 2 , and k –2 are rate constants. The reaction’s rate law for generating the product [P] is However, if we make measurement early in the reaction, the concentration of products is negligible, i.e., [P]≈ A nd we can ignore the back reaction (second term in right side of Equation  2). Then under these conditions, the reaction’s rate is, To be analytically useful we need to write Equation  4  in terms of the reactants (e.g., the concentrations of enzyme and substrate). To do this we use the  steady-state approximation , in which we assume that the concentration of  ES  remains essentially constant. Following an initial period, during which the enzyme–substrate complex first forms, the rate at which  ES   forms, I s equal to the rate at which it disappears, 2 3 4 5 6 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 5

6. where [ E]  is the enzyme’s original concentration. Combining Equations  5   and 6   gives, which we solve for the concentration of the enzyme–substrate complex, where  K m  is the  Michaelis constant . Substituting Equation 8  into Equation  4  leaves us with our final rate equation . Graph 01: Plot of Equation 9 showing limits for the analysis of substrates and enzymes in an enzyme-catalyzed chemical kinetic method of analysis. The curve in the region highlighted in red obeys equation 11 and the curve in the area highlighted in green follows Equation 10. 7 8 9 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 6

7. For high substrate concentrations, where [S] ≫K m , Equation  9  simplifies to, where  V max is the maximum rate for the catalyzed reaction. Under these conditions the reaction is zero-order in substrate and we can use  V max to calculate the enzyme’s concentration, typically using a variable-time method. At lower substrate concentrations, where [S]≪ K m , Equation 9   becomes, The reaction is now  first-order in substrate, and we can use the rate of the reaction to determine the substrate’s concentration by a fixed-time method. The Michaelis constant  K m is the substrate concentration at which the reaction rate is at half-maximum, and is an inverse measure of the substrate's affinity for the enzyme—as a small K m  indicates high affinity, meaning that the rate will approach  V max  more quickly. The value of  K m is dependent on both the enzyme and the substrate, as well as conditions such as temperature and pH . 10 11 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 7

8. From the last two terms in Equation  11 , we can express  V max in terms of a  turnover number  ( kcat ): where  [E]  is the enzyme concentration and  kcat  is the turnover number, defined as the maximum number of substrate molecules converted to product per enzyme molecule per second. Hence, the turnover number is defined as the maximum number of chemical conversions of substrate molecules per second that a single catalytic site will execute for a given enzyme concentration [ E] o . 12 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 8

9. Kinetically Perfect Enzymes Efficient. Specificity constant K cat / K m - 10 8 to 10 9 M -1 S -1 . Table 01: Some Kinetically Prefect Enzymes. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 9

10. Advantages of Kinetically Perfect Enzymes w.r.t. Biological Systems. Triose Phosphate Isomerase Acetylcholinesterase Superoxide Dismutase 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 10

11. Triose Phosphate Isomerase Fig 03: Structure of TPI enzyme. Fig 04: Reaction catalysed by TPI enzyme. A crucial enzyme involved in the glycolytic pathway. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 11

12. Acetylcholinesterase Fig 05: Structure of acetylcholinesterase enzyme. Fig 06: Reaction catalysed by acetylcholinesterase enzyme. A crucial enzyme involved in nerve impulse transmission. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 12

13. Superoxide Dismutase Fig 07: Structure of SOD enzyme. Fig 08: Reaction catalysed by SOD enzyme. A crucial enzyme involved in destruction of superoxide radicals. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 13

14. Conclusion Very important. Inability to function as kinetically perfect enzymes leads to severe toxicities in the body. Deficiencies or malfunctioning of these enzymes lead to many abnormalities such as, a ffected individuals experience low levels of circulating red blood cells due to premature destruction of red blood cells (hemolytic anemia) and severe, progressive neurological symptoms ( for TPI), neurodegenerative disorders (for AChE ),  familial amyotrophic lateral sclerosis a motor neuron disease (for SOD) etc. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 14

15. References N S Punekar , Enzymes: Catalysis, Kinetics & Mechanisms. Springer Nature Singapore. Ltd, 2018, 560 pp. Berg J M, Tymoczko J L, Stryer L, Biochemistry, 5 th Edition, W H Freeman & Company & Sumona . Inc , 2002, 1514pp. https:// chem.libretexts.org/Courses/University_of_California_Davis/UCD_Chem_107B%3A_Physical_Chemistry_for_Life_Scientists/Chapters/3%3A_Enzyme_Kinetics/3.2%3A_The_Equations_of_Enzyme_Kinetics https:// en.wikipedia.org 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 15

16. Acknowledgements I would like to thank the department of Molecular Biology for providing me this opportunity to present my seminar. I also t hank Dr. Ragavendra Hegade Katte for his valuable guidance throughout the preparation of my seminar. Thank you. 5/24/2020 Kinetically Perfect Enzymes. Shryli K S 16