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A Kinetic Analysis of the Reaction Catalysed by (Hydroxymethyl)bilane Synthase
Author(s) -
Niemann Annette C.,
Hädener Alfons,
Matzinger Peter K.
Publication year - 1994
Publication title -
helvetica chimica acta
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.74
H-Index - 82
eISSN - 1522-2675
pISSN - 0018-019X
DOI - 10.1002/hlca.19940770711
Subject(s) - chemistry , hydroxymethyl , substrate (aquarium) , steady state (chemistry) , stereochemistry , enzyme , amino acid , elution , kinetics , chromatography , organic chemistry , biochemistry , oceanography , physics , quantum mechanics , geology
(Hydroxymethyl)bilane synthase (HMBS) catalyses the conversion of porphobilinogen ( 2 ) into the (hydroxymethyl)bilane derivative 3 , a linear tetrapyrrolic intermediate in the biosynthesis of haem, chlorophyll, and related pigments. The conversion involves the sequential formation of four intermediate covalent enzyme‐substrate complexes, before the product is released. We analysed the pre‐steady‐state kinetics of the formation of the complexes, taking advantage of their remarkable chemical stability allowing chromatographic separation. The experimental approach involved the generation of the complexes while HMBS was immobilised on an anion‐exchange column. A solution being 0.2 K m in substrate was pumped through the column during a time interval which was varied to sample the pre‐steady‐state period. Then, the enzyme and enzyme‐substrate complexes were eluted and their proportions evaluated. A computer simulation of the pre‐steady‐state time course, in combination with a χ 2 fitting to the experimental data, allowed the specificity constants k cat / K m for the individual steps of the process to be derived. By repeating the analysis with variants of HMBS in which specific amino acids were replaced by others, we demonstrated that it is possible to trace the consequences of amino‐acid replacements down to the individual steps of the reaction sequence. Since the positions of the amino acids concerned in the three‐dimensional structure were known, detailed structure‐function relationships become evident in this way.