The Enzymatic Reaction and Mechanism of Lactate Dehydrogenase

Name : ANWAR Khairil

Student ID : 292240512
Lecture : Enzymology 1

Lactate Dehydrogenase (LDH) is an enzyme which plays important role of the anaerobic
metabolic pathway [1]. LDH catalyzes the reversible reaction between pyruvate and lactate. It
belongs to the oxidoreductase group and presents in almost all body tissues. Oxidoreductases
are the first major group of enzymes that catalyze the exchange of electrons between two
molecules. The electron shift takes by hydride transfer, proton-hydrogen extraction, oxygen
insertion and these enzymes usually employ NAD+ and NADP as cofactors [2].
LDH is an intracellular enzyme that is widely distributed at high levels in many different
tissues and utilizes glucose for energy during anaerobic respiration. It also helps to overcome
a temporary oxygen debt in the form of accumulated lactate, which is later discharged by the
re-oxidation to pyruvate. Isoenzymes are predominantly distributed in the tissue specific
manner. LDH-1 and LDH-2 are predominantly present in cardiac muscles, kidney, and
erythrocytes [3].
The combinations of LDH-A and LDH-B subunits generate five major isoforms of this
enzyme, with the size of molecular weight of approximately 35 kDa respectively. According
to the IUPAC recommendation, these five isoforms are numbered as LDH-1 to LDH-5.
Isoenzyme expression of LDH varies between different organs and tissues other than the heart
and muscle, depending on oxygen accessibility, environmental condition, substrate, product,
and coenzyme concentration. Even though in such varied environment, all the isoforms
catalyze the same reversible conversion of pyruvate to lactate with regeneration of NAD+,
required for continued formation of ATP from glycolysis [4].
LDH catalyzes the synchronized inter-convertion of pyruvate to lactate and NADH to
+
NAD and enhances the speed of reaction by 14 times. The chemical reaction begins by
transferring a hydride ion from NADH to pyruvate at its C2 carbon. The molecular mechanism
comprises the binding of NADH to the enzymes as a first step. Many residues at the active site
are involved in this binding. Once NADH is bound, it facilitates the binding of lactate, through
an interaction between the NADH ring and the LDH residues [1]. A hydride is rapidly
transferred in both directions, resulting in the formation of the two tertiary complexes, which
are LDH-NAD+-lactate and LDH-NADH-pyruvate [5]. Afterwards, pyruvate is dissociation of
NADH and NAD+ proves to be the rate-limiting step in this reaction, and the final conversion
of pyruvate to lactate leading to the regeneration of NAD+ is thermodynamically favored in the
reaction [1]. The reversible reaction catalyzed by lactate dehydrogenase (LDH) is described as
follows:
 Figure 1. The reversible reaction catalyzed lactate dehydrogenase (LDH)
The reversible reaction is catalyzed by LDH as follows: L-lactate + NAD+ ⇌ Pyruvate +
NADH + H+. The reaction equilibrium supports the reduction of pyruvate to lactate and the
equilibrium constant (K) is 2.8 ×10-5 mol1 as determined at 25 ˚C (pH 7.0) [6]. For the forward
reaction (Lactate → Pyruvate), substrate inhibition by lactate is less than as compared to the
product, pyruvate concentration. Several methods, such as fluorescence, difference
spectroscopy, and ultracentrifugation, have shown that the tetrameric form of LDH possesses
four separate and independent binding sites for NADH, NAD+, and substrates. The monomeric
form of the enzyme is inactive and lactate, as well as pyruvate, does not bind to the apoenzyme
[4].
Kinetic and equilibrium binding assays exhibited that the compulsory order of binding
sequence is followed by this enzyme (LDH), first to coenzyme and then substrate. During the
forward reaction (Lactate → Pyruvate), the limiting step in the oxidation of lactate at substrate
saturation is the rate of dissociation of NADH from binary complex. In reverse reaction
(Pyruvate → Lactate), the rate-limiting step is either associated with the redox reaction or with
the dissociation of the enzyme-NAD+ complex [7].
The important residues of LDH active site and their specific roles in reaction mechanisms
have been discovered through various experimental procedures. Different types of substrate
analogs and inhibitors have helped in indentifying the reaction mechanism of LDH. By the use
of oxamate, it obviously showed the reaction mechanism at the active site and the role of
important residues [4]. At the active site, some of the amino acid residues, such as Arg-171,
His-195, Arg-109, Asp-168, Val-138 play an important role [8]. During the reaction process,
Arg-171 binds and orientates the substrate, and His-195 acts as a proton donor. And then, His-
195 gets activated by Arg-109 to stabilize the transition state. The carboxamide side-chain of
cofactor orientation is carried out by Val-138, while Asp-168 conducts a role in modulation of
pKa value of His-195 [9]. Other residues in neighborhood, Gln-102 and Thr-246 are very
important in substrate discrimination, while Ile-250 keeps the hydrophobic environment of the
nicotinamide ring [4].
LDH activity is dependant on the metabolic switch to anaerobic respiration. LDH is
modulated by three types of regulations, which are allosteric modulation, substrate-level
regulation, and transcriptional regulation. The relative availability and concentration of
substrates regulate the activity of LDH. The enzyme becomes more active during extreme
muscular activity when there is an increase in substrates. The demand for ATP compared to
aerobic ATP supply causes the accumulation of ADP, AMP, and Pi [1].
To conclude, I state clearly that LDH plays a great metabolic role due to its ubiquitous
nature and different isoenzyme forms. It is one of the most important enzymes to study for
isoenzyme switching, different cellular locations, multi-organ sites and varied substrate
specificity.
 References

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dehydrogenase displays absolute stereospecificity at C4 of the nicotinamide ring. J. Am.
Chem. Soc., 110, 3695-3697.