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New assay method based on Raman spectroscopy for enzymes reacting with gaseous substrates
Author(s) -
KawaharaNakagawa Yuka,
Nishikawa Koji,
Nakashima Satoru,
Inoue Shota,
Ohta Takehiro,
Ogura Takashi,
Shigeta Yasuteru,
Fukutani Katsuyuki,
Yagi Tatsuhiko,
Higuchi Yoshiki
Publication year - 2019
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.3569
Subject(s) - chemistry , hydrogenase , substrate (aquarium) , raman spectroscopy , cuvette , spectroscopy , catalysis , enzyme catalysis , redox , active site , reaction rate constant , analytical chemistry (journal) , desulfovibrio vulgaris , chemical reaction , reversible reaction , electron transfer , kinetics , photochemistry , inorganic chemistry , chromatography , organic chemistry , oceanography , physics , quantum mechanics , biology , bacteria , optics , genetics , geology
Abstract Enzyme activity is typically assayed by quantitatively measuring the initial and final concentrations of the substrates and/or products over a defined time period. For enzymatic reactions involving gaseous substrates, the substrate concentrations can be estimated either directly by gas chromatography or mass spectrometry, or indirectly by absorption spectroscopy, if the catalytic reactions involve electron transfer with electron mediators that exhibit redox‐dependent spectral changes. We have developed a new assay system for measuring the time course of enzymatic reactions involving gaseous substrates based on Raman spectroscopy. This system permits continuous monitoring of the gas composition in the reaction cuvette in a non‐invasive manner over a prolonged time period. We have applied this system to the kinetic study of the [NiFe] hydrogenase from Desulfovibrio vulgaris Miyazaki F. This enzyme physiologically catalyzes the reversible oxidation of H 2 and also possesses the nonphysiological functions of H/D exchange and nuclear spin isomer conversion reactions. The proposed system has the additional advantage of enabling us to measure all of the hydrogenase‐mediated reactions simultaneously. Using the proposed system, we confirmed that H 2 (the fully exchanged product) is concomitantly produced alongside HD by the H/D exchange reaction in the D 2 /H 2 O system. Based on a kinetic model, the ratio of the rate constants of the H/D exchange reaction ( k ) at the active site and product release rate ( k out ) was estimated to be 1.9 ± 0.2. The proposed assay method based on Raman spectroscopy can be applied to the investigation of other enzymes involving gaseous substrates.