Direct Measurement of Enzyme Activity with Infrared Spectroscopy

A direct approach to enzyme activity measurements is presented. Vibrational spectroscopy can monitor the progress of enzymatic reactions because the vibrational spectrum of substrates and products usually differs. This is demonstrated by the example of ATP hydrolysis by Ca2+-ATPase: The substrate concentration can be followed using the infrared absorption of the α- and β-PO2 − phosphate groups of ATP, and the product concentration can be followed using the P03 2- absorption of Pi and of the fl-phosphate of ADP. The results of the infrared spectroscopic measurement of ATPase activity and of an independent activity assay agree very well. The main advantage of the infrared method is that it observes the reaction of interest directly—that is, no activity assay that converts the progress of the reaction into an observable quantity is required.

[1]  H. Murata,et al.  Interaction of adenosine 5'-triphosphate with Mg2+: vibrational study of coordination sites by use of 18O-labeled triphosphates , 1988 .

[2]  A. Barth,et al.  Substrate binding and enzyme function investigated by infrared spectroscopy , 2000, FEBS letters.

[3]  W. Mäntele,et al.  Time-resolved Infrared Spectroscopy of the Ca2+-ATPase , 1996, The Journal of Biological Chemistry.

[4]  W. Hasselbach.  Calcium-activated ATPase of the sarcoplasmic reticulum membranes , 1981 .

[5]  C. Wharton,et al.  A stopped-flow apparatus for infrared spectroscopy of aqueous solutions. , 1995, The Biochemical journal.

[6]  W. Hasselbach.  Chapter 7 Calcium-activated ATPase of the sarcoplasmic reticulum membranes , 1981 .

[7]  J. Andersen,et al.  Structure-function relationships of the calcium binding sites of the sarcoplasmic reticulum Ca(2+)-ATPase. , 1998, Acta physiologica Scandinavica. Supplementum.

[8]  D. Trentham,et al.  Properties and Uses of Photoreactive Caged Compounds , 1989 .

[9]  S. Wiberley,et al.  Introduction to infrared and Raman spectroscopy , 1965 .

[10]  J. Knowles,et al.  beta-Lactamase proceeds via an acyl-enzyme intermediate. Interaction of the Escherichia coli RTEM enzyme with cefoxitin. , 1980, Biochemistry.

[11]  K. Hauser,et al.  Photochemical Release of ATP from "Caged ATP" Studied by Time-Resolved Infrared Spectroscopy , 1995 .

[12]  G. Inesi,et al.  Kinetic Regulation of Catalytic and Transport Activities in Sarcoplasmic Reticulum ATPase , 1985 .

[13]  L. Herbette,et al.  A new approach to time-resolved studies of ATP-requiring biological systems; laser flash photolysis of caged ATP. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[14]  Bernhard Lendl,et al.  Time-Resolved FT-IR Spectroscopy of Chemical Reactions in Solution by Fast Diffusion-Based Mixing in a Micromachined Flow Cell , 2001 .

[15]  D. McIntosh,et al.  Portrait of a P-type pump , 2000, Nature Structural Biology.

[16]  D. Moss,et al.  Stopped flow system for FTIR difference spectroscopy of biological macromolecules , 1999 .

[17]  M. Shigekawa,et al.  Reaction mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. ATP hydrolysis with CaATP as a substrate and role of divalent cation. , 1983, The Journal of biological chemistry.

[18]  M. J. Gradwell,et al.  Time-Resolved Infrared Spectroscopy of Intermediates and Products from Photolysis of 1-(2-Nitrophenyl)ethyl Phosphates: Reaction of the 2-Nitrosoacetophenone Byproduct with Thiols , 1997 .

[19]  J. Andersen.  Monomer-oligomer equilibrium of sarcoplasmic reticulum Ca-ATPase and the role of subunit interaction in the Ca2+ pump mechanism. , 1989, Biochimica et biophysica acta.

[20]  Anthony N. Martonosi,et al.  The Enzymes of Biological Membranes , 1985, Springer US.

[21]  L. de Meis,et al.  Acetyl phosphate as substrate for Ca 2+ uptake in skeletal muscle microsomes. Inhibition by alkali ions. , 1971, The Journal of biological chemistry.

[22]  J. Kaplan,et al.  Rapid photolytic release of adenosine 5'-triphosphate from a protected analogue: utilization by the Na:K pump of human red blood cell ghosts. , 1978, Biochemistry.