The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis.
The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent d-lactate dehydrogenase (d-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. d-iLDH was specific for d-lactate and it was able to reduce quinones of different redox potential values. Oxalate and l-lactate were mixed-type inhibitors of d-iLDH. Mitochondrial l-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both l-iLDH and d-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that l-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD(+)-dependent LDH specific for the generation of d- and l-lactate (d-nLDH and l-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by d-nLDH was inhibited by its two products; the d-lactate oxidation was 40-fold lower than forward reaction. l-lactate oxidation by l-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K(+) or NH(4)(+). Interestingly, membrane-bound l- and d-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble d-nLDH has been identified in bacteria and some yeast. Also, FBP-activated l-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed.
Jasso-Chávez
Ricardo
R
Departamento de Bioquímica, Instituto Nacional de Cardiología, Juan Badiano #1, Col. Sección XVI, Tlalpan, México, D. F. 14080, México. rjassoch@aol.com
García-Cano
Israel
I
Marín-Hernández
Alvaro
A
Mendoza-Cózatl
David
D
Rendón
Juan Luis
JL
Moreno-Sánchez
Rafael
R
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D004734:Energy Metabolism; D005056:Euglena gracilis / Q000201:enzymology*; D019143:Evolution, Molecular; D007700:Kinetics; D045022:Lactate Dehydrogenases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D019344:Lactic Acid / Q000737:chemistry / Q000378:metabolism; D015394:Molecular Structure
D001426:Bacterial Proteins; D019344:Lactic Acid; D045022:Lactate Dehydrogenases; C071195:D-lactate dehydrogenase