scholarly journals Protein Conformational Landscapes and Catalysis. Influence of Active Site Conformations in the Reaction Catalyzed by L-Lactate Dehydrogenase

ACS Catalysis ◽  
2015 ◽  
Vol 5 (2) ◽  
pp. 1172-1185 ◽  
Author(s):  
Katarzyna Świderek ◽  
Iñaki Tuñón ◽  
Sergio Martí ◽  
Vicent Moliner
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site

scholarly journals Comparison of the activity and conformation changes of lactate dehydrogenase H4 during denaturation by guanidinium chloride

1991 ◽  
Vol 277 (1) ◽  
pp. 207-211 ◽  
Author(s):  
Y Z Ma ◽  
C L Tsou
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Cross Linking ◽  
Native Conformation ◽  
Guanidinium Chloride ◽  
Original Activity ◽  
Limited Region ◽  
Low Concentrations ◽  
Two Stages ◽  
Inhibited Enzyme

The inactivation and unfolding of lactate dehydrogenase (LDH) during denaturation by guanidinium chloride (GuHCl) under diverse conditions have been compared. Unfolding of the native conformation, as monitored by fluorescence and c.d. measurements, occurs in two stages with increasing GuHCl concentrations, and the inactivation approximately coincides with, but slightly precedes, the first stage of unfolding. The enzyme is inhibited to about 60-70% of its original activity by cross-linking with glutaraldehyde or in the presence of 1 M-(NH4)2SO4, with its conformation stabilized as shown by the requirement for higher GuHCl concentrations to bring about both inactivation and unfolding. Low concentrations of GuHCl (0.2-0.4 M) activate the cross-linked and the (NH4)2SO4-inhibited enzyme back to the level of the native enzyme. For the enzyme stabilized by (NH4)2SO4 or by cross-linking with glutaraldehyde, inactivation occurs at a markedly lower GuHCl concentration than that required to bring about its first stage of unfolding. It is concluded that the active site of LDH is situated in a limited region relatively fragile in conformation as compared with the molecule as a whole. The GuHCl activation of LDH stabilized in (NH4)2SO4 or by cross-linking with glutaraldehyde suggests that this fragility and consequently flexibility of the active site is required for its catalytic activity.

Active-Site Heterogeneity of Lactate Dehydrogenase

ACS Catalysis ◽  
2019 ◽  
Vol 9 (5) ◽  
pp. 4236-4246 ◽  
Author(s):  
He Yin ◽  
Hui Li ◽  
Adam Grofe ◽  
Jiali Gao
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Site Heterogeneity

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 Modification of pig heart lactate dehydrogenase with methyl methanethiosulphonate to produce an enzyme with altered catalytic activity

1977 ◽  
Vol 161 (3) ◽  
pp. 643-651 ◽  
Author(s):  
D P Bloxham ◽  
D C Wilton
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Thiol Group ◽  
Dilute Solution ◽  
Substrate Analogues ◽  
Steady State Kinetics ◽  
Active Methyl ◽  
Catalytically Active ◽  
Modified Enzyme ◽  
Formation Of Complexes

Methyl methanethiosulphonate was used to produce a modification of the essential thiol group in lactate dehydrogenase which leaves the enzyme catalytically active. Methyl methanethiosulphonate produced a progressive inhibition of enzyme activity, with 2mM-pyruvate and 0.14mM-NADH as substrates, which ceased once the enzyme had lost 70-90% of its activity. In contrast, with 10mM-lactate and 0.4mM-NAD+ as substrates the enzyme was virtually completely inhibited. The observed inhibition was critically dependent on the chosen substrate concentration, since methanethiolation with methyl methanethiosulphonate resulted in a large decrease in affinity for pyruvate. At 0.14mM-NADH, methanethiolation increased the apparent KmPyr from from 40micronM for the control enzyme to 12mM for the modified enzyme. Steady-state kinetics showed that there was not a statistically significant change in either KmNADH or KsNADH. At saturating NADH and pyruvate concentrations, the Vmax. was virtually unaffected for the methanethiolated enzyme. However, a decrease in Vmax. was observed when the modified enzyme was incubated in dilute solution. The modification of lactate dehydrogenase by methyl methanethiosulphonate involved the active site, since inhibition was completely prevented by substrate-analogue pairs such as NADH and oxamate or NAD+ and oxalate. The formation of complexes between methanethiolated lactate dehydrogenase and substrates or substrate analogues can also be shown by re-activation experiments. The methanethiolated enzyme was re-activated in a time-dependent reaction by dithiothreitol and this was prevented by oxamate, by NADH and by NADH plus oxamate in increasing order of effectiveness. The results of this work are interpreted in terms of a role for the essential thiol group in the binding of substrates.

scholarly journals The mechanism of adduct formation between NAD+ and pyruvate bound to pig heart lactate dehydrogenase

1979 ◽  
Vol 177 (3) ◽  
pp. 951-957 ◽  
Author(s):  
D C Wilton
Keyword(s):  
Lactate Dehydrogenase ◽  
Dissociation Constant ◽  
Active Site ◽  
Adduct Formation ◽  
Rabbit Muscle ◽  
Muscle Lactate

1. The rate of adduct formation between NAD+ and enol-pyruvate at the active site of lactate dehydrogenase is determined by the rate of enolization of pyruvate in solution. 2. The proportion of enol-pyruvate solutions is less than 0.01%. 3. The overall dissociation constant of adduct formation is less than 5 × 10(-8) M for pig heart lactate dehydrogenase at pH 7.0. 4. The unusual kinetics for adduct formation previously observed in the case of rabbit muscle lactate dehydrogenase [Griffin & Criddle (1970) Biochemistry 9, 1195–1205] may be attributed to the concentration of enol-pyruvate in solution being considerably less than the concentration of enzyme.

scholarly journals Structural characterization of the apo form and NADH binary complex of human lactate dehydrogenase

2014 ◽  
Vol 70 (5) ◽  
pp. 1484-1490 ◽  
Author(s):  
Sally Dempster ◽  
Stephen Harper ◽  
John E. Moses ◽  
Ingrid Dreveny
Keyword(s):  
Lactate Dehydrogenase ◽  
Conformational Changes ◽  
Active Site ◽  
Anaerobic Respiration ◽  
Crystal Form ◽  
Binary Complex ◽  
Cofactor Binding ◽  
Key Enzyme ◽  
Crystallization Conditions

Lactate dehydrogenase A (LDH-A) is a key enzyme in anaerobic respiration that is predominantly found in skeletal muscle and catalyses the reversible conversion of pyruvate to lactate in the presence of NADH. LDH-A is overexpressed in many tumours and has therefore emerged as an attractive target for anticancer drug discovery. Crystal structures of human LDH-A in the presence of inhibitors have been described, but currently no structures of the apo or binary NADH-bound forms are available for any mammalian LDH-A. Here, the apo structure of human LDH-A was solved at a resolution of 2.1 Å in space groupP4122. The active-site loop adopts an open conformation and the packing and crystallization conditions suggest that the crystal form is suitable for soaking experiments. The soaking potential was assessed with the cofactor NADH, which yielded a ligand-bound crystal structure in the absence of any inhibitors. The structures show that NADH binding induces small conformational changes in the active-site loop and an adjacent helix. A comparison with other eukaryotic apo LDH structures reveals the conservation of intra-loop interactions. The structures provide novel insight into cofactor binding and provide the foundation for soaking experiments with fragments and inhibitors.

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 Identification of New Structural Fragments for the Design of Lactate Dehydrogenase A Inhibitors

Acta Naturae ◽  
2016 ◽  
Vol 8 (3) ◽  
pp. 118-122
Author(s):  
D. K. Nilov ◽  
A. V. Kulikov ◽  
E. A. Prokhorova ◽  
V. K. Švedas
Keyword(s):  
Lactate Dehydrogenase ◽  
Active Site ◽  
Structural Modification ◽  
Experimental Testing ◽  
Sulfo Group ◽  
Inhibitory Effect ◽  
Structural Elements ◽  
Molecular Fragments ◽  
Closed Conformation ◽  
Guanidinium Group

Human lactate dehydrogenase A plays an important role in the glucose metabolism of tumor cells and constitutes an attractive target for chemotherapy. Molecular fragments able to bind in the active site of this enzyme and form hydrogen bonds with the Arg168 guanidinium group, as well as additional interactions with the loop 96-111 in the closed conformation, have been identified by virtual screening of sulfonates and experimental testing of their inhibitory effect. The sulfo group can occupy a similar position as the carboxyl group of the substrate and its structural analogs, whereas the benzothiazole group attached via a linker can be located in the coenzyme (NADH) binding site. Thus, the value of merging individual structural elements of the inhibitor by a linker was demonstrated and ways of further structural modification for the design of more effective inhibitors of lactate dehydrogenase A were established.

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