ATP formation onset lag and post-illumination phosphorylation initiated with single-turnover flashes. I. An assay using luciferin-luciferase luminescence

1988 ◽  
Vol 20 (1) ◽  
pp. 85-106 ◽  
Author(s):  
William A. Beard ◽  
Richard A. Dilley
Keyword(s):  
Single Turnover ◽  
Atp Formation

A Dynamic Substrate is Required for MhuD-catalyzed Degradation of Heme to Mycobilin

2021 ◽  
Author(s):  
Biswash Thakuri ◽  
Bruce O'Rourke ◽  
Amanda Graves ◽  
Matthew Liptak
Keyword(s):  
Dynamic Equilibrium ◽  
Ionization Mass ◽  
Esi Ms ◽  
Enzymatic Mechanism ◽  
Single Turnover ◽  
Rate Limiting Step ◽  
Planar Substrate ◽  
Out Of Plane ◽  
Substrate Conformation ◽  
Rate Limiting

The non-canoncial heme oxygenase MhuD from <i>Mycobacterium tuberculosis</i> binds a heme substrate that adopts a dynamic equilibrium between planar and out-of-plane ruffled conformations. MhuD degrades this substrate to an unusual mycobilin product via successive monooxygenation and dioxygenation reactions. This article establishes a causal relationship between heme substrate dynamics and MhuD-catalyzed heme degradation resulting in a refined enzymatic mechanism. UV/Vis absorption (Abs) and electrospray ionization mass spectrometry (ESI-MS) data demonstrated that a second-sphere substitution favoring population of the ruffled heme conformation changed the rate-limiting step of the reaction resulting in a measurable build-up of the monooxygenated meso-hydroxyheme intermediate. In addition, UV/Vis Abs and ESI-MS data for a second-sphere variant that favored the planar substrate conformation showed that this change altered the enzymatic mechanism resulting in an alpha-biliverdin product. Single-turnover kinetic analyses for three MhuD variants revealed that the rate of heme monooxygenation depends upon the population of the ruffled substrate conformation. These kinetic analyses also revealed that the rate of meso-hydroxyheme dioxygenation by MhuD depends upon the population of the planar substrate conformation. Thus, the ruffled haem conformation supports rapid heme monooxygenation by MhuD, but further oxygenation to the mycobilin product is inhibited. In contrast, the planar substrate conformation exhibits altered heme monooxygenation regiospecificity followed by rapid oxygenation of meso-hydroxyheme. Altogether, these data yielded a refined enzymatic mechanism for MhuD where access to both substrate conformations is needed for rapid incorporation of three oxygen atoms into heme yielding mycobilin.<br>

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