scholarly journals Mechanism of action of porphobilinogen deaminase. The participation of stable enzyme substrate covalent intermediates between porphobilinogen and the porphobilinogen deaminase from Rhodopseudomonas spheroides

1981 ◽  
Vol 195 (1) ◽  
pp. 177-181 ◽  
Author(s):  
P M Jordan ◽  
A Berry
Keyword(s):  
Mechanism Of Action ◽  
Catalytic Cycle ◽  
Porphobilinogen Deaminase ◽  
Enzyme Substrate ◽  
Enzyme Intermediates ◽  
Rhodopseudomonas Spheroides ◽  
Covalently Bound

Highly stable labelled complexes are formed between porphobilinogen deaminase and stoicheiometric amounts of [14C]porphobilinogen. On completion of the catalytic cycle by the addition of excess of substrate, the complexes yield labelled product and display all the properties expected from covalently bound enzyme intermediates involved in the deaminase catalytic sequence.

scholarly journals Modification of hydroxymethylbilane synthase (porphobilinogen deaminase) by pyridoxal 5′-phosphate. Demonstration of an essential lysine residue

1984 ◽  
Vol 222 (1) ◽  
pp. 93-102 ◽  
Author(s):  
G J Hart ◽  
F J Leeper ◽  
A R Battersby
Keyword(s):  
Euglena Gracilis ◽  
Enzymic Activity ◽  
Lysine Residue ◽  
Porphobilinogen Deaminase ◽  
Amino Group ◽  
Spectroscopic Evidence ◽  
Lysine Residues ◽  
Modified Enzyme ◽  
Rhodopseudomonas Spheroides ◽  
Covalently Bound

When hydroxymethylbilane synthase (porphobilinogen deaminase) from Euglena gracilis is incubated with pyridoxal 5′-phosphate at pH 7.0 and 0 degree C, it rapidly loses part of its activity. The proportion of activity that remains decreases as the concentration of the modifier increases up to approx. 2mM, above which no further significant inactivation occurs. Dialysis of the partly inactivated enzyme restores its activity, whereas reduction with NaBH4 makes the inactivation permanent. The maximum inactivation achievable from one cycle of the treatment with pyridoxal 5′-phosphate, then with borohydride, is 53 +/- 5%; taking this modified enzyme through second and third cycles causes further loss of activity. The enzyme from Rhodopseudomonas spheroides behaves similarly, but there are quantitative differences. Spectroscopic evidence indicates that the inactivation procedure modifies lysine residues, and labelling studies show that epsilon-N-pyridoxyl-L-lysine is a product when permanently inactivated enzyme is completely hydrolysed. Several lysine residues per molecule of the E. gracilis enzyme are modified by the treatment with pyridoxal 5′-phosphate and borohydride, but only one appears to be essential for enzymic activity, since porphobilinogen protects the enzyme against inactivation and then one fewer lysine residue per molecule of enzyme is affected. It is suggested that, during the biosynthesis of hydroxymethylbilane, the first porphobilinogen unit is covalently bound to the enzyme through the epsilon-amino group of the essential lysine.

Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation

2003 ◽  
Vol 31 (3) ◽  
pp. 731-735 ◽  
Author(s):  
P.M. Shoolingin-Jordan ◽  
A. Al-Dbass ◽  
L.A. McNeill ◽  
M. Sarwar ◽  
D. Butler
Keyword(s):  
Recombinant Proteins ◽  
Acute Intermittent Porphyria ◽  
Catalytic Cycle ◽  
Porphobilinogen Deaminase ◽  
Catalytic Residue ◽  
Enzyme Substrate ◽  
Arginine Residues ◽  
Chain Polymerization ◽  
Low Activity ◽  
Insight Into

Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.

scholarly journals A novel approach to distinguish between enzyme mechanisms: quasi-steady-state kinetic analysis of the prostaglandin H synthase peroxidase reaction

2003 ◽  
Vol 372 (3) ◽  
pp. 713-724 ◽  
Author(s):  
Peter V. VRZHESHCH ◽  
Elena A. BATANOVA ◽  
Alevtina T. MEVKH ◽  
Sergei D. VARFOLOMEEV ◽  
Irina G. GAZARYAN ◽  
...  
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Catalytic Cycle ◽  
Prostaglandin H Synthase ◽  
Compound I ◽  
Novel Approach ◽  
Steady State Kinetic ◽  
Enzyme Intermediates ◽  
Prostaglandin H ◽  
State Kinetic

A method of analysis for steady-state kinetic data has been developed that allows relationships between key partial reactions in the catalytic cycle of a functioning enzyme to be determined. The novel approach is based on a concept of scalar and vector ‘kinetic connectivities’ between enzyme intermediates in an arbitrary enzyme mechanism. The criterion for the agreement between experimental data and a proposed kinetic model is formulated as the kinetic connectivity of intermediate forms of the enzyme. This concept has advantages over conventional approaches and is better able to describe the complex kinetic behaviour of prostaglandin H synthase (PGHS) when catalysing the oxidation of adrenaline by H2O2. To interpret the experimental data for PGHS, a generalized model for multi-substrate enzyme reactions was developed with provision for irreversible enzyme inactivation. This model showed that two enzyme intermediates must undergo inactivation during the catalytic cycle. These forms are proposed to be PGHS compound I and a compound I–adrenaline complex.

Properties of Chlorophyllase from Capsicum annuum L. Fruits

2001 ◽  
Vol 56 (11-12) ◽  
pp. 1015-1021 ◽  
Author(s):  
Dámaso Hornero-Méndez ◽  
María Isabel Mínguez-Mosquera
Keyword(s):  
Substrate Specificity ◽  
Capsicum Annuum ◽  
Mechanism Of Action ◽  
Substrate Recognition ◽  
Substrate Inhibition ◽  
Inhibitory Effect ◽  
Hplc Separation ◽  
Substrate Complex ◽  
Enzyme Substrate

Abstract The in vitro properties of semi-purified chlorophyllase (chlorophyll-chlorophyllido hy­drolase, EC 3.1.1.14) from Capsicum annuum fruits have been studied. The enzym e showed an optimum of activity at pH 8.5 and 50 °C. Substrate specificity was studied for chlorophyll (Chi) a, Chi b, pheophytin (Phe) a and Phe b, with Km values of 10.70, 4.04, 2.67 and 6.37 μᴍ respectively. Substrate inhibition was found for Phe b at concentrations higher than 5 μᴍ. Chlorophyllase action on Chi a' and Chi b' was also studied but no hydrolysis was observed, suggesting that the mechanism of action depends on the configuration at C-132 in the chloro­ phyll molecule, with the enzyme acting only on compounds with R132 stereochemistry. The effect of various metals (Mg2+, Hg2+, Cu2+, Zn2+, Co , Fe2+ and Fe3+) was also investigated, and a general inhibitory effect was found, this being more marked for Hg2+ and Fe2+. Func­tional groups such as -SH and -S-S-seem ed to participate in the formation o f the enzyme-substrate complex. Chelating ion and the carbonyl group at C3 appeared to be important in substrate recognition by the enzyme. The method for measuring Chlase activity, including HPLC separation of substrate and product, has been optimized.

scholarly journals Discovery that the assembly of the dipyrromethane cofactor of porphobilinogen deaminase holoenzyme proceeds initially by the reaction of preuroporphyrinogen with the apoenzyme

1996 ◽  
Vol 316 (2) ◽  
pp. 373-376 ◽  
Author(s):  
Peter M. SHOOLINGIN-JORDAN ◽  
Martin J. WARREN ◽  
Sarah J. AWAN
Keyword(s):  
Escherichia Coli ◽  
Catalytic Cycle ◽  
Intermediate Complex ◽  
Porphobilinogen Deaminase ◽  
Assembly Process

The assembly process of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase holoenzyme is initiated by the reaction of the porphobilinogen deaminase apoenzyme with preuroporphyrinogen. The resulting enzyme-bound tetrapyrrole (bilane) is equivalent to the holoenzyme intermediate complex ES2 and yields the dipyrromethane cofactor by reactions of the normal catalytic cycle. These observations indicate that preuroporphyrinogen, rather than porphobilinogen, is the preferred precursor for the dipyrromethane cofactor and explain the existence of the D84A and D84N deaminase mutants as catalytically inactive ES2 complexes.

Sign in / Sign up

Export Citation Format

Share Document