scholarly journals Transient kinetics of the reaction catalysed by magnesium protoporphyrin IX methyltransferase

2004 ◽  
Vol 382 (3) ◽  
pp. 1009-1013 ◽  
Author(s):  
Mark SHEPHERD ◽  
C. Neil HUNTER
Keyword(s):  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Tryptophan Fluorescence ◽  
Lag Phase ◽  
Transient Kinetics ◽  
Methyl Group ◽  
Synechocystis Pcc6803 ◽  
Propionate Group ◽  
Tryptophan Fluorescence Quenching ◽  
Flow Techniques

Magnesium protoporphyrin IX methyltransferase (ChlM), an enzyme in the chlorophyll biosynthetic pathway, catalyses the transfer of a methyl group to magnesium protoporphyrin IX (MgP) to form magnesium protoporphyrin IX monomethyl ester (MgPME). S-Adenosyl-L-methionine is the other substrate, from which a methyl group is transferred to the propionate group on ring C of the porphyrin macrocycle. Stopped-flow techniques were used to characterize the binding of porphyrin substrate to ChlM from Synechocystis PCC6803 by monitoring tryptophan fluorescence quenching on a millisecond timescale. We concluded that a rapid binding step is preceded by a slower isomerization of the enzyme. Quenched-flow techniques have been employed to characterize subsequent partial reactions in the catalytic mechanism. A lag phase has been identified that has been attributed to the formation of an intermediate. Our results provide a greater understanding of this catalytic process which controls the relative concentrations of MgP and MgPME, both of which are implicated in signalling between the plastid and nucleus in plants.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Biochemical Study of Multiple CheY Response Regulators of the Chemotactic Pathway of Rhodobacter sphaeroides

2004 ◽  
Vol 186 (15) ◽  
pp. 5172-5177 ◽  
Author(s):  
Axelle Ferré ◽  
Javier de la Mora ◽  
Teresa Ballado ◽  
Laura Camarena ◽  
Georges Dreyfus
Keyword(s):  
Fluorescence Quenching ◽  
Rhodobacter Sphaeroides ◽  
Biochemical Study ◽  
Response Regulator ◽  
Tryptophan Fluorescence ◽  
Flagellar Motor ◽  
Acetyl Phosphate ◽  
Response Regulators ◽  
Tryptophan Fluorescence Quenching

ABSTRACT The six copies of the response regulator CheY from Rhodobacter sphaeroides bind to the switch protein FliM. Phosphorylation by acetyl phosphate (AcP) was detected by tryptophan fluorescence quenching in three of the four CheYs that contain this residue. Autophosphorylation with Ac32P was observed in five CheY proteins. We also show that all of the cheY genes are expressed simultaneously; therefore, in vivo all of the CheY proteins could bind to FliM to control the chemotactic response. Consequently, we hypothesize that in this complex chemotactic system, the binding of some CheY proteins to FliM, does not necessarily imply switching of the flagellar motor.

scholarly journals Isolation of two N-monosubstituted protoporphyrins, bearing either the whole drug or a methyl group on the pyrrole nitrogen atom, from liver of mice given griseofulvin

1991 ◽  
Vol 274 (3) ◽  
pp. 843-848 ◽  
Author(s):  
A E Holley ◽  
Y Frater ◽  
A H Gibbs ◽  
F De Matteis ◽  
J H Lamb ◽  
...  
Keyword(s):  
Nitrogen Atom ◽  
Protoporphyrin Ix ◽  
Secondary Product ◽  
Structural Isomers ◽  
Green Pigment ◽  
Methyl Group ◽  
Group 4 ◽  
Pyrrole Nitrogen

1. A hepatic green pigment with inhibitory properties towards the enzyme ferrochelatase has been isolated from the liver of mice treated with griseofulvin and identified as N-methylprotoporphyrin. 2. All four structural isomers of N-methylprotoporphyrin have been demonstrated to be present, NA, where ring A of protoporphyrin IX is N-methylated, being the predominant isomer. 3. In addition to N-methylprotoporphyrin, a second green pigment, present in far greater amounts, was also isolated from the liver of griseofulvin-treated mice. This second green pigment is also an N-monosubstituted protoporphyrin, but in this case the substituent on the pyrrole nitrogen atom appears to be intact griseofulvin rather than a methyl group. 4. The fragmentation of this adduct in tandem m.s. studies suggests that griseofulvin is bound to the pyrrole nitrogen through one of its carbon atoms and further suggests that N-methylprotoporphyrin may arise as a secondary product from the major griseofulvin pigment.

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