In the 4 years since Hammes (87) reviewed, in this journal, the application of flow and relaxation techniques to the study of primary processes in chemical reactions these methods have been applied to a wide range of biochemical problems. These applications have been facilitated by advances in stopped flow and temperature jump instrumentation, especially with respect to the range of physical properties that may be observed during a reaction, and the availability of instruments.

https://science.sciencemag.org/content/sci/170/3955/273.full.pdf

Kinetic studies on the ES-complex formation of p-hydroxybenzoate hydroxylase using the stopped-flow method.

Measurement of fast biochemical reactions.

1. The first metabolic step in the biodegradation of 4-hydroxypyridine by an Agrobacterium sp. was hydroxylation to form pyridine-3,4-diol. 2. Extracts required 1mol of O(2) and 1mol of NADH or NADPH for the conversion of 4-hydroxypyridine into pyridine-3,4-diol, suggesting that the enzyme responsible, 4-hydroxypyridine-3-hydroxylase, was a mixed function mono-oxygenase. 3. After treatment with acidic (NH(4))(2)SO(4) the enzyme required FAD for activity; FMN and riboflavin would not substitute for FAD. 4. The rate of anaerobic reduction of FAD by NAD(P)H was increased more than tenfold in the presence of 4-hydroxypyridine, suggesting that the mechanism of hydroxylation was similar to that of other aromatic hydroxylases which are of the mono-oxygenase type. 5. The partially purified enzyme was extremely specific for its heterocyclic substrate but would utilize either NADH or NADPH. 6. 4-Hydroxypyridine-3-hydroxylase was strongly inhibited by high substrate concentration (above 0.5mm) especially below pH7.5. 8. The inflexion at pH8.4 in a pK(m) versus pH plot, together with strong inhibition by p-chloromercuribenzoate, suggested a role for thiol groups in substrate binding.

/pdf/microbial-metabolism-of-the-pyridine-ring-the-hydroxylation-2e0gn1ko12.pdf

Microbial metabolism of the pyridine ring. The hydroxylation of 4-hydroxypyridine to pyridine-3,4-diol (3,4-dihydroxypyridine) by 4-hydroxypyridine-3-hydroxylase.

Kinetic studies on the ES-complex formation of p-hydroxybenzoate hydroxylase using the stopped-flow method.

Citations

Measurement of fast biochemical reactions.

Microbial metabolism of the pyridine ring. The hydroxylation of 4-hydroxypyridine to pyridine-3,4-diol (3,4-dihydroxypyridine) by 4-hydroxypyridine-3-hydroxylase.

Characterization of a mixing device adapted to the kinetics of a specific enzyme: the horse liver alcohol dehydrogenase model

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