1994
DOI: 10.1002/hlca.19940770711
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A Kinetic Analysis of the Reaction Catalysed by (Hydroxymethyl)bilane Synthase

Abstract: (Hydroxymethy1)bilane synthase (HMBS) catalyses the conversion of porphobilinogen (2) into the (hydroxymethy1)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 chr… Show more

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Cited by 7 publications
(8 citation statements)
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“…As expected, ES 4 was not detected at all, as this intermediate is short-lived and is rapidly hydrolyzed into the linear HMB product ( Warren and Jordan, 1988 ). This observed distribution is consistent with results from earlier studies ( Bustad et al., 2013 ; Niemann et al., 1994 ; Shoolingin-Jordan et al., 2003 ), demonstrating the ability of ESI-FT-ICR MS to separate and directly identify different co-existing enzyme-substrate intermediates through intact protein mass analysis. In addition, the mass accuracy was high enough to directly distinguish between the reduced (DPM) and the oxidized (dipyrromethene) form of the cofactor (i.e., 2 Da mass difference in a 40 kDa protein), and our results conclusively indicated that the DPM cofactor existed exclusively in its reduced DPM form ( Figure S1 ).…”
Section: Resultssupporting
confidence: 91%
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“…As expected, ES 4 was not detected at all, as this intermediate is short-lived and is rapidly hydrolyzed into the linear HMB product ( Warren and Jordan, 1988 ). This observed distribution is consistent with results from earlier studies ( Bustad et al., 2013 ; Niemann et al., 1994 ; Shoolingin-Jordan et al., 2003 ), demonstrating the ability of ESI-FT-ICR MS to separate and directly identify different co-existing enzyme-substrate intermediates through intact protein mass analysis. In addition, the mass accuracy was high enough to directly distinguish between the reduced (DPM) and the oxidized (dipyrromethene) form of the cofactor (i.e., 2 Da mass difference in a 40 kDa protein), and our results conclusively indicated that the DPM cofactor existed exclusively in its reduced DPM form ( Figure S1 ).…”
Section: Resultssupporting
confidence: 91%
“…The ES 2 intermediate was the most abundant, whereas ES was only found in a very small amount and ES 4 was not detected at all. ES is kinetically less stable than ES 2 or ES 3 , and ES 2 accumulates during the reaction, in agreement with a slow rate of the ES 2 →ES 3 step ( Niemann et al., 1994 ; Warren and Jordan, 1988 ). The presence of the apoenzyme in wt-hPBGD is remarkable and has not been observed before in any enzyme preparation is from prokaryote expression, as it is assumed to be unstable and less structurally compact than E holo ( Awan et al., 1997 ; Scott et al., 1989 ).…”
Section: Discussionsupporting
confidence: 52%
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“…The danger then is that the crystal structures are artefactual or, if not exactly that, then at least not natural. A mutant such as K59Q (Niemann et al, 1994) does not fully stop the enzyme at this second stage; rather, it slows the process down, which I think is a better approach to the natural state of the enzyme. In this case the X-ray diffraction experiment has to be sufficiently quick in its measurements to capture this accumulating population (Helliwell et al, 1998).…”
Section: Discussionmentioning
confidence: 99%
“…Also, there are possibilities, via protein engineering (e.g. see Niemann et al 1994), and cryotechniqes (Petsko, 1992 and references therein) to control the rates of enzyme reactions to some extent. The justification of time-resolved methods comes when structural information from 'static' crystallography of one or a series of protein structures still cannot give key details.…”
Section: Io Dynamical Studies Of Proteins In Crystalsmentioning
confidence: 99%