Kinetic properties of 5-carboxymethyl-2-hydroxymuconate semialdehyde dehydrogenase from Escherichia coli |
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| Authors: | J M Alonso A Garrido-Pertierra |
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| Affiliation: | 1. Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia in Katowice, Jagiellonska 28, 40-032 Katowice, Poland;2. Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, 30-239, Cracow, Poland;1. Departamento de Bioquímica, Facultad de Química, Universidad Nacional Autónoma de México, México D. F. 04510, Mexico;2. Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, México D. F. 04510, Mexico;1. Department of Chemical Engineering, University of California Santa Barbara, Santa Barbara, CA 93106, United States;2. Science and Technology Research Center, Inc., Mitsubishi Rayon Group, Yokohama, Kanagawa 227-8502, Japan;1. Department of Biosystems and Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 136-713, Republic of Korea;2. Department of Clinical Laboratory Science, College of Health Sciences, Catholic University of Pusan, Busan 609-757, Republic of Korea;1. G.K. Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia;2. Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia;3. Institute of Ecology and Genetics of Microorganisms, Ural Branch, Russian Academy of Sciences, Perm, Russia |
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| Abstract: | Kinetic properties of purified 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMSA) dehydrogenase (EC 1.2.1.-) in the 4-hydroxyphenylacetate meta-cleavage pathway from Escherichia coli have been studied. The temperature--activity relationship for the enzyme from 27 to 45 degrees C showed an Arrhenius plot with an inflexion at 36 degrees C. When 5-carboxymethyl-2-hydroxymuconic semialdehyde and NAD were used as variable substrates, the double reciprocal plots were all linear and the lines intersected at one point below the horizontal axis, suggesting that a sequential mechanism is operating. From the replots of intercepts and slopes against reciprocal substrate concentrations were calculated Km (CHMSA) = 9.0 +/- 1.02 microM, Km (NAD) = 29.1 +/- 4.65 microM and the value for the dissociation constant of enzyme--NAD complex = 6.3 +/- 1.21 microM. ATP and the product of the reaction (NADH) acted as competitive inhibitors of the enzyme with respect to NAD. Apparent Ki values, estimated from Dixon plots, were 25.0 +/- 3.5 and 88.0 +/- 22.1 microM for NADH and ATP, respectively. |
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