A method for determining kinetic parameters at high enzyme concentrations.
A graphical method is described which allows determination of kinetic parameters when substrate, inhibitor or activator concentrations must be in the vicinity of the enzyme concentration and a significant fraction of ligand is bound. Velocity is measured at several ligand: enzyme ratios at two or more enzyme concentrations. Results are obtained in terms of free and bound ligand corresponding to particular velocities. The relationship between velocity and bound and free ligand may then be analysed by any desired plotting technique. Preknowledge of the reaction mechanism or experimental determination of Vmax. is not required. The relationship between ligand bound and enzyme activity need not be linear and the method is equally suitable for analysing co-operative as well as simple kinetics. Application of the method is demonstrated by analysis of the inhibition of fructose, 1,6-bisphosphatase by AMP.
13 references, click to expand
-
Kidney fructose 1,6-diphosphatase. Purification and studies on desensitization to adenosine 5'-monophosphate inhibition.Arch Biochem Biophys. 1967 Nov;122(2):393-9 PMID: 6066246
-
Functional consequences of modification of kidney fructose 1,6-diphosphatase by pyridoxal 5'-phosphate.J Biol Chem. 1968 Sep 25;243(18):4923-6 PMID: 4301229
-
Electrostatic methods for measuring the binding of ionic ligands to proteins.Biochemistry. 1971 Sep 14;10(19):3564-9 PMID: 5169542
-
Selective alteration of the regulatory properties of fructose 1,6-diphosphatase by modification with pyridoxal 5'-phosphate.Biochemistry. 1972 May 9;11(10):1798-803 PMID: 4337193
-
Method for measuring the binding of small molecules to proteins from binding-induced alterations of physical-chemical properties.Biochemistry. 1972 Aug 29;11(18):3493-8 PMID: 5066444
-
A linear equation that describes the steady-state kinetics of enzymes and subcellular particles interacting with tightly bound inhibitors.Biochem J. 1972 Apr;127(2):321-33 PMID: 4263188
-
The graphical determination of K m and K i .Biochem J. 1972 Aug;129(1):197-202 PMID: 4630451
-
Steady-state enzyme kinetics with high-affinity substrates or inhibitors. A statistical treatment of dose-response curves.Biochem J. 1973 Sep;135(1):101-7 PMID: 4204669
-
Tight-binding inhibitors--III. A new approach for the determination of competition between tight-binding inhibitors and substrates--inhibition of adenosine deaminase by coformycin.Biochem Pharmacol. 1976 Dec 15;25(24):2695-702 PMID: 1008893
-
In vivo and in vitro phosphorylation of rat liver fructose-1,6-bisphosphatase.Proc Natl Acad Sci U S A. 1977 Oct;74(10):4615-9 PMID: 200922
-
The determination of binding parameters when the total and free substrate concentrations are not approximately equal.Biochem J. 1979 Jun 1;179(3):697-700 PMID: 475775
-
Evaluation of methods for estimating the dissociation constant of tight binding enzyme inhibitors.J Biol Chem. 1979 Dec 10;254(23):12104-9 PMID: 500698
-
ALLOSTERIC INHIBITION OF RAT LIVER FRUCTOSE 1,6-DIPHOSPHATASE BY ADENOSINE 5'-MONOPHOSPHATE.J Biol Chem. 1965 Feb;240:651-62 PMID: 14275118
Contact
No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong
Qilu Normal University · Genelibs Bioinformatics Lab
750 Shunhua Rd, Jinan
2F, Bldg F, University Science Park
Tel: 0531-88819269
WeChat Official Account
Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.
Business Email
E-mail: product@genelibs.com
Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2
