Non-equilibrium Binding Energy Determined Using Alpha-amylase Catalysed Amylolysis of Gelatinised Starch as a Probable Generalisable Model and Importance

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Ikechukwu I. Udema


Objectives: This research was undertaken to determine the non–equilibrium binding energy by calculation after substituting experimental data into derived equations, present its role distinct from energy associated with activated enzyme–substrate (ES) complex and ultimately elucidate the importance of binding energies.

Background: There are overwhelming pieces of evidence in the literature that binding interaction is essential for the ultimate transformation of a substrate, inhibition of vital enzymes of pathogens, covid-19 in particular. Intrinsic binding energy herein referred to as non–equilibrium binding energy and energy associated with activated ES are seen to be chemical in origin. Much attention seemed not to be given to theoretical approach to the determination of non–equilibrium binding energy.

Methods: Experimental approach (Bernfeld method of enzyme assay) and calculational.

Results and Discussion: The non–equilibrium translational (2.691–2.726 kJ/mol) and total electrostatic energies (2.755-3.154 kJ/mol) were > than the thermal energy at 310.15 k. The interfacial distance between the bullet and target molecule was expectedly very short; the range was between 6.672 and 7.570 exp (- 12) m. This was attributed to the interaction between charged enzyme and weakly polar substrate.

Conclusion: The equations of non–equilibrium and translational energies were derivable. The binding interaction serves to fix the bullet molecule on or into the target (supra) molecule before the commencement of transition state formation. The non–equilibrium binding interactions of the bullet (drugs, substrate, etc) and target (receptors e.g. enzymes, pathogens such as Covid–19, Plasmodium etc) and the ultimate complex are likely to be stabilised against the thermal energy in furtherance of enzymatic and drug action since the electrostatic interaction energy is higher than thermal energy.

Gelatinised insoluble potato starch, human salivary alpha–amylase (EC, non– equilibrium interaction energy, total translational energy, total electrostatic interaction energy.

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How to Cite
Udema, I. I. (2020). Non-equilibrium Binding Energy Determined Using Alpha-amylase Catalysed Amylolysis of Gelatinised Starch as a Probable Generalisable Model and Importance. Asian Journal of Chemical Sciences, 8(3), 9-23.
Original Research Article


Pan R, Xue-Jing Zhang X-J, Zhang Z-J, Zhou Y, Wei-Xi Tian W-X, He R-Q. Conformation impact on subsequent reactions with substrates. J. Biol. Chem. 2010;85(30):22950–22956.

Koshland DE (Jr). Application of enzyme specificity to protein synthesis. 1958;44: 98–104.

Fischer E. Influence of the configuration on the effect of the enzyme. Ber. Dtsch. Chem. Ges. 1894;27:2985–2993.

Yao QZ, Tian M, Tsou CL. Comparison of the rates of inactivation and conformational changes of creatine kinase during urea denaturation. Biochemistry. 1984;23(12): 2740–2744.

Davies DR, Cohen GH. Interactions of protein antigens with antibodies. Proc. Natl. Acad. Sci. USA. 1996;93(1):7–12.

Sundberg EJ, Mariuzza RA. Luxury accommodations: The expanding role of structural plasticity in protein – protein interactions. Structure. 2000;8(7):137–142.

Bakan A Bahar I. The intrinsic dynamics of enzymes plays a dominant role in determining the structural changes induced upon inhibitor binding. Proc. Nat. Acad. Sci. USA. 2009;106(34):14349–14354.

Swift RV, McCammon JA. Substrate induced population shifts and stochastic gating in the PBCV – 1 mRNA capping enzyme. J. Am. Chem. Soc. 2009;131: 5126–5133.

Weikl TR, von Deuster C. Selected fit versus induced fit protein binding: Kinetic differences and mutational analysis. Proteins. 2009;75(1):104–110.

Sullivan SM, Holyoak T. Enzymes with lid-gated active sites must operate by an induced fit mechanism instead of conformational selection. Proc. Natl. Acad. Sci. USA. 2008;105(37):13829–13834.

Okazaki K, Takada S. Dynamic energy landscape view of coupled binding and protein conformational change: Induced fit versus population shift mechanism. Proc. Natl. Acad. Sci. USA. 2008;105(32): 11182–11187.

D’Amico S, Marx J–C, Gerday C, Feller G. Activity–stability relationships in extremophilic enzymes. J. Biol. Chem. 2003;276(10):7891–7896.

Reuveni S, Urbakhc M, Klafterc J. Role of substrate unbinding in Michaelis–Menten enzymatic reactions. Proc. Natl. Acad. Sci. USA. 2014;1-6.

Mahdian S, Ebrahim–Habibi A, Zarrabi M. Drug repurposing using computational methods to identify therapeutic options for COVID-19. J. Diabetes Metab. Disord. 2020;1–9.

Hu X, Cai X, Song X, Li C, Zhao J, Luo W, et al. Possible SARS–coronavirus 2 inhibitor revealed by simulated molecular docking to viral main protease and host toll-like receptor. Future Virol. 2020;1– 10.

Choudhary S, Malik YS, Tomar S. Identification of sars–cov–2 cell entry inhibitors by drug repurposing using in silico structure-based virtual screening approach. Front. Immunol. 2020;11(1664): 1–14.

Hertel KJ, Peracchi A, Uhlenbeck OC, Herschlag D. Use of intrinsic binding energy for catalysis by an RNA enzyme. Proc. Natl. Acad. Sci. USA. Biochemistry. 1997;94:8497–8502.

Amyes TL, Malabanan MM, Zhai X, Reyes AC, Richard JP. Enzyme activation through the utilisation of intrinsic dianion binding energy. Protein Eng. Des. Sel. 2017;30(3):159–167.

Szklarz GD, Paulsen MD. Molecular modeling of cytochrome P450 1A1: Enzyme–substrate interactions and substrate binding affinities. J. Biomol. Struct. & Dyn. 2002;20(2):155–162.

Singh M, Singh S, Inamuddin, Asiri AM. IFT and friccohesity study of formulation, wetting, dewetting of liquid systems using oscosurvismeter. J. Mol. Liq. 2017;244:7–18.

Udema II. Determination of translational velocity of reaction mixture components: Effect on the rate of reaction. Adv. Biochem. 2016;4(6):84–93.

Udema II, Onigbinde AO. The state of proteins notwithstanding, translational velocity is vital for their function. Asian J. Res. Biochem. 2019;5(3):1–17.

Chetty R, Singh M. In-vitro interaction of cerium oxide nanoparticles with hemoglobin, insulin and dsDNA at 310.15 K: Physicochemical, spectroscopic and in silico study. Int. J. Biol. Macromol. 2020;156:1022–1044.

Udema II. The key to effective catalytic action is pre-catalytic site activity preceding enzyme– substrate complex formation. Adv. Res. 2017;9(3):1–12.

Udema II. Determination of molar mass and its relationship with free energy of activation: A case study on human salivary alpha–amylase. J. Sci. Res. Reports. 2016;12(5):1–11.

Neurath H, Cooper GR. The diffusion constant of tomato bushy stunt virus. J. Biol. Chem. 1940;135:455–162.

Tomasik P. Specific physical and chemical properties of potato starch. Food (Special Issue 1). 2008;45–56.

Gao D, Chundawat SPS, Sethi A, Balan V, Gnanakaran S, Dale BE. Increased enzyme binding to substrate is not necessary for more efficient cellulose hydrolysis. Proc. Natl. Acad. Sci. 2013; 110(27):10922–10927.

Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: Biochemical properties and novel applications. Microbiol. Mol. Biol. Rev. 2006;70(2):283–295.

Jencks WP. Binding energy, specificity, and enzymic catalysis: The circe effect. Adv. Enzymol. Relat. Areas Mol. Biol. 1975;43:219–410.

Milstien S, Cohen LA. Rate acceleration by stereopopulation control: Models for enzyme action. Proc. Natl. Acad. Sci. USA. 1970;67:1143–1147.

Storm DR, Koshland DE, Jr. A source for the special catalytic power of enzymes Orbital steering. Proc. Natl. Acad. Sci. USA. 1970;66:445–452.

Page MI, Jencks WP. Entropic contributions to rate accelerations in enzymic and intramolecular reactions and the chelate effect. Proc. Natl. Acad. Sci. USA. 1971;68:16781683.

Cannon WR, Singleton SF, Benkovic SJ. A perspective on biological catalysis. Nat. Struct. Biol. 1996;3:821–833.

Dewar MJ. New ideas about enzyme reactions. Enzyme. 1986;36:8–20.

Warshel A, Aqvist J, Creighton S. Enzymes work by solvation substitution rather than by desolvation. Proc. Natl. Acad. Sci. USA. 1989;86:5820–5824.

Lockhart DJ, Kim PS. Electrostatic screening of charge and dipole interactions with the helix backbone. Science. 1993;260:198–202.

Jencks WP. Utilisation of binding energy and coupling rules for active transport and other coupled vectorial processes. Methods Enzymol. 1989;171:145–164.

Jencks WP. Economics of enzyme catalysis. Cold Spring Harb. Symp. Quant. Biol. 1987;52:65–73.

Pauling L. Molecular architecture and biological reactions. Chem Eng News. 1946;24:1375–1377.

Schwans JP, Kraut DA, Herschlag D. Determining the catalytic role of remote substrate binding interactions in ketosteroid isomerase. Biochemistry. 2009;106(34):14271–14275.

Makawana D, Singh M. A new dendrimer series: Synthesis, free radical scavenging and protein binding studies. RSC Adv. 2020;10:21914–21932.

Abdul Kadhim AH, Hadi NR, Abdulhussein M, Abdulhussein M, Zamil ST, Zamil ST. Preprocessing of the candidate antiviral drugs against COVID-19 in models of SARS cov2 targets. Prensa Med. Argent. 2020;106(2):240–249.

Schleinkofer K, Wang T, Wade RC. Molecular docking. In: Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine. Springer, Berlin, Heidelberg; 2006.

Pérez S, Tvaroška I. Carbohydrate–protein interactions: Molecular modeling insights. Adv Carbohydr Chem Biochem. 2014;71: 9–136.

Lim–Wilby M, Morris GM. Molecular docking. Methods Mol. Biol. 2008;443: 365–82.

Monika G, Punam G, Sarbjot S, Gupta GD. An overview on molecular docking. Int. J. Drug. Dev. & Res. 2010;2(2):219–231.

Rohs R, Bloch I, Sklenar H, Shakked Z. Molecular flexibility in ab–initio drug docking to DNA: Binding-site and binding-mode transitions in all-atom Monte Carlo simulations. Nucl. Acids Res. 2005;33: 7048–7057.

Guedes IA, de Magalhães CS, Dardenne LE. Receptor-ligand molecular docking. Biophys. Rev. 2014;6:75–87.

Udema II. Effect of salts and organic osmolytes is due to conservative forces. Asian J. Appl. Chem. Res. 2020;5(1):1–17.

Gopich IV, Szabo A. Diffusion modifies the connectivity of kinetic schemes for multisite binding and catalysis. Proc. Natl. Acad. Sci. USA. 2013;110(49):19784–19789.

Lund M. Electrostatic interactions in and between biomolecules. Lund University. Ph.D Thesis; 2006.