Lactate dehydrogenase amino acid composition

Enzymes which exist in multiple forms within a single species of organism or even in a single cell are called isoenzymes or isozymes. Such multiple forms can be detected and separated by gel electrophoresis of cell extracts. Since they are coded by different genes, they differ in amino acid composition and thus in their isoelectric pH values. Lactate dehydrogenase is an example for the isoenzymes which occur as five different forms in the tissues of the human and other vertebrates. All the five isozymes catalyze the same reaction. 

The amino acid compositions of a number of lactate dehydrogenases (H4, M4, and X4) are given in Table II. Chemical modification of one cysteine per subunit (122) results in loss of activity (see Section II,B). Hence, cysteine in LDH has received particular attention. No disulfide bridges have been reported for LDH (123). The enzyme does not contain any metals 53,124)-... 

Amino Acid Composition of Some Lactate Dehydrogenases... 

Similarly, if one compares the form of the enzyme lactate dehydrogenase found in heart muscle to the type found in skeletal muscle, one can see small differences in amino acid composition. These differences in turn affect the reaction catalyzed by this enzyme, the conversion of pyruvate to lactate. The heart type has a high Kf, or a low afiinity for pyruvate, and the muscle type has a low K, or a high afiinily for pyruvate. This means that the pyruvate win be preferentially converted to lactate in the muscle but will be preferentially used for aerobic metabolism in the heart, rather than being converted to lactate. These conclusions are consistent with the known biology and metabolism of these two tissues. 

Reflect and Apply The M and H subunits of lactate dehydrogenase have very similar sizes and shapes but differ in amino acid composition. If the only difference between the two were that the H subunit had a glutamic acid in a position where the M subunit had a serine, how would the five isozymes of LDH separate on electrophoresis using a gel at pH 8.6 (See Chapter 5 for details on electrophoresis.)... 

Isozymes are oligomeric enzymes that have slightly different amino acid compositions in different organs. Lactate dehydrogenase is an example, as is phosphofructokinase. 

A typical spectrum of individual lactate dehydrogenases (LDH) of muscles after electrophoresis on starch gel is shown in Fig. 81 (Markert and Appella, 1963). These five fractions, as mentioned previously, are determined by (he existence of two genetic loci for synthesis of two types of protein subunits, specific as regards amino acid composition and antigenicity, designated A and B, and by the presence of a tetramer structure of the principal protein (AAAA-LDH-5 AAAB-LDH-4 AABB-LDH-3 ABBB-LDH-2 BBBB-LDH-1). 

An example of an enzyme which has different isoenzyme forms is lactate dehydrogenase (LDH) which catalyzes the reversible conversion of pyruvate into lactate in the presence of the coenzyme NADH (see above). LDH is a tetramer of two different types of subunits, called H and M, which have small differences in amino acid sequence. The two subunits can combine randomly with each other, forming five isoenzymes that have the compositions H4, H3M, H2M2, HM3 and M4. The five isoenzymes can be resolved electrophoretically (see Topic B8). M subunits predominate in skeletal muscle and liver, whereas H subunits predominate in the heart. H4 and H3M isoenzymes are found predominantly in the heart and red blood cells H2M2 is found predominantly in the brain and kidney while HM3 and M4 are found predominantly in the liver and skeletal muscle. Thus, the isoenzyme pattern is characteristic of a particular tissue, a factor which is of immense diagnostic importance in medicine. Myocardial infarction, infectious hepatitis and muscle diseases involve cell death of the affected tissue, with release of the cell contents into the blood. As LDH is a soluble, cytosolic protein it is readily released in these conditions. Under normal circumstances there is little LDH in the blood. Therefore the pattern of LDH isoenzymes in the blood is indicative of the tissue that released the isoenzymes and so can be used to diagnose a condition, such as a myocardial infarction, and to monitor the progress of treatment. 

---