Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG)

Kalina Ranguelova, Javier Suarez, Leonid Metlitsky, Shengwei Yu, Shelly Zev Brejt, Sidney Zelig Brejt, Lin Zhao, Johannes Schelvis, Richard S. Magliozzo

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Abstract

The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.

Original languageEnglish
Pages (from-to)12583-12592
Number of pages10
JournalBiochemistry
Volume47
Issue number47
DOIs
StatePublished - 25 Nov 2008

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Mycobacterium tuberculosis
Catalase
Peroxidase
Heme
Ligands
Water
Enzymes
Peroxides
Cyanides
Potassium Cyanide
Optical flows
Isoniazid
Pathogens
Titration
Purification
Paramagnetic resonance
Virulence
Spectrum Analysis
Tuberculosis
Demonstrations

Cite this

Ranguelova, K., Suarez, J., Metlitsky, L., Yu, S., Brejt, S. Z., Brejt, S. Z., ... Magliozzo, R. S. (2008). Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG). Biochemistry, 47(47), 12583-12592. https://doi.org/10.1021/bi801511u
Ranguelova, Kalina ; Suarez, Javier ; Metlitsky, Leonid ; Yu, Shengwei ; Brejt, Shelly Zev ; Brejt, Sidney Zelig ; Zhao, Lin ; Schelvis, Johannes ; Magliozzo, Richard S. / Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG). In: Biochemistry. 2008 ; Vol. 47, No. 47. pp. 12583-12592.
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title = "Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG)",
abstract = "The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.",
author = "Kalina Ranguelova and Javier Suarez and Leonid Metlitsky and Shengwei Yu and Brejt, {Shelly Zev} and Brejt, {Sidney Zelig} and Lin Zhao and Johannes Schelvis and Magliozzo, {Richard S.}",
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Ranguelova, K, Suarez, J, Metlitsky, L, Yu, S, Brejt, SZ, Brejt, SZ, Zhao, L, Schelvis, J & Magliozzo, RS 2008, 'Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG)', Biochemistry, vol. 47, no. 47, pp. 12583-12592. https://doi.org/10.1021/bi801511u

Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG). / Ranguelova, Kalina; Suarez, Javier; Metlitsky, Leonid; Yu, Shengwei; Brejt, Shelly Zev; Brejt, Sidney Zelig; Zhao, Lin; Schelvis, Johannes; Magliozzo, Richard S.

In: Biochemistry, Vol. 47, No. 47, 25.11.2008, p. 12583-12592.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Impact of distal side water and residue 315 on ligand binding to ferric Mycobacterium tuberculosis catalase - Peroxidase (KatG)

AU - Ranguelova, Kalina

AU - Suarez, Javier

AU - Metlitsky, Leonid

AU - Yu, Shengwei

AU - Brejt, Shelly Zev

AU - Brejt, Sidney Zelig

AU - Zhao, Lin

AU - Schelvis, Johannes

AU - Magliozzo, Richard S.

PY - 2008/11/25

Y1 - 2008/11/25

N2 - The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.

AB - The catalase-peroxidase (KatG) of Mycobacterium tuberculosis (Mtb) is important for the virulence of this pathogen and also is responsible for activation of isoniazid (INH), an antibiotic in use for over 50 years in the first line treatment against tuberculosis infection. Overexpressed Mtb KatG contains a heterogeneous population of heme species that present distinct spectroscopic properties and, as described here, functional properties. A six-coordinate (6-c) heme species that accumulates in the resting enzyme after purification is defined as a unique structure containing weakly associated water on the heme distal side. We present the unexpected finding that this form of the enzyme, generally present as a minority species along with five-coordinate (5-c) enzyme, is the favored reactant for ligand binding. The use of resting enzyme samples with different proportional composition of 5-c and 6-c forms, as well as the use of KatG mutants with replacements at residue 315 that have different tendencies to stabilize the 6-c form, allowed demonstration of more rapid cyanide binding and preferred peroxide binding to enzyme containing 6-c heme. Optical-stopped flow and equilibrium titrations of ferric KatG with potassium cyanide reveal complex behavior that depends in part on the amount of 6-c heme in the resting enzymes. Resonance Raman and low-temperature EPR spectroscopy clearly demonstrate favored ligand (cyanide or peroxide) binding to 6-c heme. The 5-c and 6-c enzyme forms are not in equilibrium on the time scale of the experiments. The results provide evidence for the likely participation of specific water molecule(s) in the first phases of the reaction mechanism of catalase-peroxidase enzymes.

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U2 - 10.1021/bi801511u

DO - 10.1021/bi801511u

M3 - Article

VL - 47

SP - 12583

EP - 12592

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

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