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 The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

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

Structural basis for the binding of succinate to succinyl-CoA synthetase

2016 ◽  
Vol 72 (8) ◽  
pp. 912-921 ◽  
Author(s):  
Ji Huang ◽  
Marie E. Fraser
Keyword(s):  
Citric Acid ◽  
Binding Site ◽  
Active Site ◽  
Citric Acid Cycle ◽  
Histidine Residue ◽  
Acid Cycle ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Active Site Histidine ◽  
Succinyl Coa Synthetase

Succinyl-CoA synthetase catalyzes the only step in the citric acid cycle that provides substrate-level phosphorylation. Although the binding sites for the substrates CoA, phosphate, and the nucleotides ADP and ATP or GDP and GTP have been identified, the binding site for succinate has not. To determine this binding site, pig GTP-specific succinyl-CoA synthetase was crystallized in the presence of succinate, magnesium ions and CoA, and the structure of the complex was determined by X-ray crystallography to 2.2 Å resolution. Succinate binds in the carboxy-terminal domain of the β-subunit. The succinate-binding site is near both the active-site histidine residue that is phosphorylated in the reaction and the free thiol of CoA. The carboxy-terminal domain rearranges when succinate binds, burying this active site. However, succinate is not in position for transfer of the phosphoryl group from phosphohistidine. Here, it is proposed that when the active-site histidine residue has been phosphorylated by GTP, the phosphohistidine displaces phosphate and triggers the movement of the carboxylate of succinate into position to be phosphorylated. The structure shows why succinyl-CoA synthetase is specific for succinate and does not react appreciably with citrate nor with the other C4-dicarboxylic acids of the citric acid cycle, fumarate and oxaloacetate, but shows some activity with L-malate.

scholarly journals The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

2008 ◽  
Vol 14 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Danuta M. Gillner ◽  
David L. Bienvenue ◽  
Boguslaw P. Nocek ◽  
Andrzej Joachimiak ◽  
Vincentos Zachary ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Active Site ◽  
Diaminopimelic Acid ◽  
Histidine Residues ◽  
Active Site Histidine

scholarly journals Functional and structural resilience of the active site loop in the evolution of Plasmodium lactate dehydrogenase

2018 ◽  
Author(s):  
Jacob D. Wirth ◽  
Jeffrey I. Boucher ◽  
Joseph R. Jacobowitz ◽  
Scott Classen ◽  
Douglas L. Theobald
Keyword(s):  
Plasmodium Falciparum ◽  
Lactate Dehydrogenase ◽  
Malate Dehydrogenase ◽  
Active Site ◽  
Drug Target ◽  
Insertion Sequence ◽  
High Potential ◽  
Ldh Activity

AbstractThe malarial pathogen Plasmodium falciparum (Pf) is a member of the Apicomplexa, which independently evolved a highly specific lactate dehydrogenase (LDH) from an ancestral malate dehydrogenase (MDH) via a five-residue insertion in a key active site loop. PfLDH is widely considered an attractive drug target due to its unique active site. Apicomplexan loop conservation suggests that a particular insertion sequence was required to evolve LDH specificity, and we previously showed (Boucher 2014) that a tryptophan in the insertion, W107f, is essential for activity and specificity. However, the roles of other residues in the loop are currently unknown. Here we show that PfLDH activity is remarkably resilient to radical perturbations of both loop identity and length. Thus, alternative insertions could have evolved LDH specificity as long as they contained a tryptophan in the proper location. PfLDH therefore has high potential to develop resistance to drugs that target its distinctive active site.

scholarly journals Modification of lactate oxidase with diethyl pyrocarbonate. Evidence for an active-site histidine residue

1977 ◽  
Vol 165 (2) ◽  
pp. 385-393 ◽  
Author(s):  
Choong Yee Soon ◽  
Maxwell G. Shepherd ◽  
Patrick A. Sullivan
Keyword(s):  
Enzyme Activity ◽  
Chemical Reduction ◽  
Enzyme Inactivation ◽  
Subunit Structure ◽  
Histidine Residue ◽  
Lactate Oxidase ◽  
Histidine Residues ◽  
Diethyl Pyrocarbonate ◽  
Competitive Inhibitors ◽  
Active Site Histidine

1. Diethyl pyrocarbonate inactivated l-lactate oxidase from Mycobacterium smegmatis. 2. Two histidine residues underwent ethoxycarbonylation when the enzyme was treated with sufficient reagent to abolish more than 90% of the enzyme activity, but analyses of the inactivation showed that the modification of one histidine residue was sufficient to cause the loss of enzyme activity. The rates of enzyme inactivation and histidine modification were the same. 3. Substrate and competitive inhibitors decreased the maximum extent of inactivation to a 50% loss of enzyme activity and modification was decreased from 1.9 to 0.75–1.2 histidine residues modified/molecule of FMN. 4. Treatment of the enzyme with diethyl [14C]pyrocarbonate (labelled in the carbonyl groups) confirmed that only histidine residues were modified under the conditions used and that deacylation of the ethoxycarbonylhistidine residues by hydroxylamine was concomitant with the removal of the14C label and the re-activation of the enzyme. 5. No evidence was found for modification of tryptophan, tyrosine or cysteine residues, and no difference was detected between the conformation and subunit structure of the modified and native enzyme. 6. Modification of the enzyme with diethyl pyrocarbonate did not alter the following properties: the binding of competitive inhibitors, bisulphite and substrate or the chemical reduction of the flavin group to the semiquinone or fully reduced states. The normal reduction of the flavin by lactate was, however, abolished.

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