Structure and Function of Dehydrogenases

Lactate dehydrogenase

In animals, L-lactate is constantly produced from pyruvate in a process of fermentation during

normal metabolism and exercise.

The concentration of lactate is ~1-2 mM at rest, but can rise to over 20 mM during intense

exertion.

This occurs due to metabolism in red blood cells that lack mitochondria.

Why is there any lactate at rest?

Limitations resulting from the enzyme activity that occurs in muscle fibers having a high

glycolytic capacity.

Enzyme isoforms

Functional lactate dehydrogenase are homo or hetero tetramers composed of M and H protein

subunits encoded by the LDHA and LDHB genes (~75% identity), respectively.

LDH-1 (4H) - heart and RBC

LDH-2 (3H1M) - reticuloendothelial system

LDH-3 (2H2M) - lungs

LDH-4 (1H3M) - kidneys, placenta, pancreas

LDH-5 (4M) – liver, striated muscle

S-lactate + NAD+ ↔ pyruvate + NADH

1. NAD-dependent

1. EC: 1.1.1.27 – acting on L-lactate (S-lactate)

2. EC: 1.1.1.28 – acting on D-lactate (R-lactate)

Four classes of lactate dehydrogenase

2. Cytochrome c-dependent

1. EC: 1.1.2.3 – acting on L-lactate (S-lactate)

2. EC: 1.1.2.4 – acting on D-lactate (R-lactate)

S-lactate + 2 ferricytochrome c ↔ pyruvate + 2 ferrocytochrome c + 2 H+.

Why is there a requirement of four enzymes performing the same function?

Cytochrome c-dependent LDH

PDB id: 1KBI

PDB id: 1I10

NAD-dependent LDH

In anaerobic cells, the ratio of pyruvate/lactate is much less than 1 while under aerobic

conditions the ratio of pyruvate/lactate is much greater than 1. Why?

In the absence of oxygen (anaerobic), the conversion of pyruvate to lactate is the only reaction

that can regenerate NAD+ allowing further glycolysis.

Cytochrome c-dependent LDH contains two domains: (1) Cytochrome binding and (2) FMN

binding.

The enzyme is a soluble component of the mitochondrial intermembrane space, where it

catalyses the reduction of pyruvate to lactate.

The enzyme transfers electrons resulting from

the oxidation of lactate into pyruvate directly

to cytochrome c.

L- Vs D- NAD-dependent LDH

PDB id: 3KB6PDB id: 1I10

Lactic acid biosynthesis

Reduction of pyruvate by L-LDH

Reduction of pyruvate by D-LDH

Mechanism of enzymatic action

Disease associated with lactic acid production

Lactic acid producing bacteria can grow in the mouth responsible for the tooth decay known

as caries.

Glucose vs Lactic acid

In brain metabolism, lactate is proposed to be the main source of energy metabolized by

neurons in the brain of several mammals species including humans.

Aldehyde dehydrogenase

EC: 1.2.1.3 An aldehyde + NAD+ + H2O = a carboxylate + NADH

EC: 1.2.1.4 An aldehyde + NADP+ + H2O = a carboxylate + NADPH

Locations and function

Aldehyde dehydrogenase is a polymorphic enzyme mostly found in the liver. Carboxylic acid

produced in the liver are metabolized by the body’s muscle and heart.

In addition, these enzymes are found in many other tissues of the body.

There are three different classes of these enzymes in mammals: class 1 (low Km, cytosolic),

class 2 (low Km, mitochondrial) and class 3 (high Km, such as those expressed in tumors,

stomach and cornea).

To date, nineteen ALDH genes have been identified within the human genome.

Biological unit

NAD binding site

Cys302 and Glu268 interact with the

aldehyde substrate.

Mechanism of action

RCHO + NAD+ + H2O → RCOOH + NADH + H+

Glutamate dehydrogenase

EC: 1.4.1.2 L-glutamate + H2O + NAD+ = 2-oxoglutarate + NH3 + NADH

EC: 1.4.1.4 L-glutamate + H2O + NADP+ = 2-oxoglutarate + NH3 + NADPH

GDH converts glutamate to α-ketoglutarate and vice versa and are required for urea synthesis.

These are present in most microbes and the mitochondria of eukaryotes. In animals, the

produced ammonia is usually used as a substrate in the urea cycle.

Location and function

Structure of GDH

Reaction catalyzed by GDH

Deamination of amino acids to the appropriate ketone

Mechanism of enzymatic action

Regulation of GDH

Under low blood glucose and caloric restriction, GDH activity is raised in order to increase

the amount of α-ketoglutarate which can be used in the citric acid cycle to ultimately produce

ATP.

In humans, the activity of GDH in insulin-producing β cells is monitored through ADP-

ribosylation, a covalent modification carried out by the gene SIRT4.

β cells secrete insulin in response to an increase in the ATP:ADP ratio. As amino acids are

broken down by GDH into α-ketoglutarate, this ratio rises and more insulin is secreted. SIRT4

is necessary to regulate the metabolism of amino acids as a method of controlling insulin

secretion and regulating blood glucose levels.

In microbes, the activity of GDH is controlled allosterically by the binding of ammonium

and/or rubidium ion.

Clinical application

GDH is important for distinguishing between acute viral hepatitis and acute toxic liver

necrosis or acute hypoxic liver disease, particularly in the case of liver damage with very high

aminotransferases.

In clinical trials, GDH can serve as a measurement for the safety of a drug.

Elevated blood serum GDH levels indicate liver damage. Thus, GDH can play a role in the

differential diagnosis of liver disease.