scholarly journals About the pKaof the active-site histidine in flavocytochrome b2(yeast L-lactate dehydrogenase)

1998 ◽  
Vol 7 (7) ◽  
pp. 1531-1537 ◽  
Author(s):  
K. Sudhindra Rao ◽  
Florence Lederer
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Flavocytochrome B2 ◽  
Active Site Histidine

An investigation of the contribution made by the carboxylate group of an active site histidine-aspartate couple to binding and catalysis in lactate dehydrogenase

Biochemistry ◽  
1988 ◽  
Vol 27 (5) ◽  
pp. 1617-1622 ◽  
Author(s):  
Anthony R. Clarke ◽  
Helen M. Wilks ◽  
David A. Barstow ◽  
Tony Atkinson ◽  
William N. Chia ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Carboxylate Group ◽  
Active Site Histidine

scholarly journals The carbanion of nitroethane is an inhibitor of, and not a substrate for, flavocytochrome b2 [l-(+)-lactate dehydrogenase]

1990 ◽  
Vol 266 (1) ◽  
pp. 301-304 ◽  
Author(s):  
R Genet ◽  
F Lederer
Keyword(s):  
Electron Transfer ◽  
Amino Acid ◽  
Lactate Dehydrogenase ◽  
Active Site ◽  
Competitive Inhibitor ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Flavocytochrome B2 ◽  
Enzyme Substrate

Although nitroethane does not bind to the active site of flavocytochrome b2, its anion, ethane nitronate, behaves as a competitive inhibitor, with a Ki of 2.2 mM. No electron transfer can be detected between the nitronate and the enzyme, in contrast with the observations of other workers on D-amino acid oxidase. Propionate is a competitive inhibitor, with a Ki of 28 mM. The significance of these results with respect to the proposed carbanion mechanism and the putative existence of a covalent enzyme-substrate intermediate is discussed.

scholarly journals Malate dehydrogenase of the cytosol. Ionizations of the enzyme-reduced-coenzyme complex and a comparison with lactate dehydrogenase

1978 ◽  
Vol 173 (2) ◽  
pp. 597-605 ◽  
Author(s):  
A Lodola ◽  
D M Parker ◽  
R Jeck ◽  
J J Holbrook
Keyword(s):  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Active Site ◽  
Histidine Residue ◽  
Histidine Residues ◽  
Coenzyme Binding ◽  
Active Site Histidine

1. The pH-dependencies of the binding of NADH and reduced nicotinamide–benzimidazole dinucleotide to pig heart cytoplasmic malate dehydrogenase and lactate dehydrogenase are reported. 2. Two ionizing groups were observed in the binding of both reduced coenzymes to lactate dehydrogenase. One group, with pKa in the range 6.3–6.7, is the active-site histidine residue and its deprotonation weakens binding of reduced coenzyme 3-fold. Binding of both coenzymes is decreased to zero when a second group, of pKa 8.9, deprotonates. This group is not cysteine-165.3. Only one ionization is required to characterize the binding of the two reduced coenzymes to malate dehydrogenase. The group involved appears to be the active-site histidine residue, since its ethoxycarbonylation inhibits the enzyme and abolishes binding of reduced coenzyme. Binding of either reduced coenzyme increases the pKa of the group from 6.4 to 7.4, and deprotonation of the group is accompanied by a 10-fold weakening of coenzyme binding. 4. Two reactive histidine residues were detected per malate dehydrogenase dimer. 5. A mechanism which emphasizes the homology between the two enzymes is presented.

scholarly journals Re-design of Saccharomyces cerevisiae flavocytochrome b2: introduction of l-mandelate dehydrogenase activity

1998 ◽  
Vol 333 (1) ◽  
pp. 117-120 ◽  
Author(s):  
Rhona SINCLAIR ◽  
Graeme A. REID ◽  
Stephen K. CHAPMAN
Keyword(s):  
Lactate Dehydrogenase ◽  
Dehydrogenase Activity ◽  
Active Site ◽  
Mutant Enzyme ◽  
Activity Levels ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Activity Increase ◽  
Double Mutation ◽  
Flavocytochrome B2

Flavocytochrome b2 from Saccharomyces cerevisiaeis an l-lactate dehydrogenase which exhibits only barely detectable activity levels towards another 2-hydroxyacid, l-mandelate. Using protein engineering methods we have altered the active site of flavocytochrome b2 and successfully introduced substantial mandelate dehydrogenase activity into the enzyme. Changes to Ala-198 and Leu-230 have significant effects on the ability of the enzyme to utilize l-mandelate as a substrate. The double mutation of Ala-198 → Gly and Leu-230 → Ala results in an enzyme with a kcat value (25 °C) with l-mandelate of 8.5 s-1, which represents an increase of greater than 400-fold over the wild-type enzyme. Perhaps more significantly, the mutant enzyme has a catalytic efficiency (as judged by kcat/Km values) that is 6-fold higher with l-mandelate than it is with l-lactate. Closer examination of the X-ray structure of S. cerevisiae flavocytochrome b2 led us to conclude that one of the haem propionate groups might interfere with the binding of l-mandelate at the active site of the enzyme. To test this idea, the activity with l-mandelate of the independently expressed flavodehydrogenase domain (FDH), was examined and found to be higher than that seen with the wild-type enzyme. In addition, the double mutation of Ala-198 → Gly and Leu-230 → Ala introduced into FDH produced the greatest mandelate dehydrogenase activity increase, with a kcat value more than 700-fold greater than that seen with the wild-type holoenzyme. In addition, the enzyme efficiency (kcat/Km) of this mutant enzyme was more than 20-fold greater with l-mandelate than with l-lactate. We have therefore succeeded in constructing an enzyme which is now a better mandelate dehydrogenase than a lactate dehydrogenase.

Functional replacement of yeast flavocytochrome b2 with bacterial l-lactate dehydrogenase

2010 ◽  
Vol 110 (3) ◽  
pp. 269-272 ◽  
Author(s):  
Hiroaki Sakai ◽  
Kazuhiro Nagahama ◽  
Hisataka Taguchi ◽  
Takashi Akamatsu ◽  
Shigeru Morimura ◽  
...  
Keyword(s):  
Lactate Dehydrogenase ◽  
Flavocytochrome B2 ◽  
Functional Replacement

Identification of an active site histidine in urokinase

1976 ◽  
Vol 429 (1) ◽  
pp. 252-257 ◽  
Author(s):  
Eng Bee Ong ◽  
Alan J. Johnson ◽  
Guenther Schoellmann
Keyword(s):  
Active Site ◽  
Active Site Histidine
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