Complete amino acid sequence of rat L-type pyruvate kinase deduced from the cDNA sequence

Abstract

cDNA clones, containing the entire coding region of rat L-type pyruvate kinase, were isolated and their nucleotide sequences were determined by the dideoxy-chain-termination method. The predicted coding region, which spans 543 amino acids, established the complete amino acid sequence of the L-type isozynie of pyruvate kinase for the first time. The deduced amino acid sequence ofthe L type has one phosphorylation site in its amino terminus and shows about 68% and 48% homologies with MI-type pyruvate kinase of chicken and yeast pyruvate kinase respectively. Domain A exhibits higher homology than domains B and C. The residues in the active site of the L-type enzyme of rats, lying between domains B and A2, are rather different from those of the M,-type enzyme of chickens, but other residues constituting the active site are identical with those of the chicken M , type except for one amino acid substitution.Four isozymes of pyruvate kinase, an important glycolytic enzyme, have been found in mammals. These are named the L type, R type, M I type and M 2 type and differ in enzymatic properties as well as in regulation of gene expression [ l -41. We have purified the four isozymes from rat tissues and characterized them [S]. They all consist of four identical subunits of about 60000 D a and all but the M1 type are allosterically regulated. The L type is very similar, but not identical, to the R type in amino acid composition, peptide fingerprint patterns and immunological properties [I, 6, 71.Both isozymes are also phosphorylated by cyclic-AMP-dependent protein kinase, which results in their inactivation [8,, in fact, showed that the two isozymes are encoded by the same gene but have distinct messenger RNAs. A similar relation between the M I and Mz types has been reported [14, 151. However, little information is available on the structures of protein and gene of these isozymes except for the M1 typeThe M 2 type is the only isozyme detected in early fetal tissues [2,19]. In adults, pyruvate kinase isozymes show tissuespecific distributions [2,4, 191; the L type is the major isozyme in liver, the M I type is the main form in muscle, heart and brain, and the R type is detected only in red cells. Transition from the fetal to adult type occurs in the late fetal to early postnatal period. Thus, expression of pyruvate kinase isozymes is tissue-specific and is regulated developmentally. This regulation is probably at the level of transcription. In addition, expression of the hepatic L-type pyruvate kinase gene is regulated by insulin and glucagon [20, 211.

cDNA cloning of human R-type pyruvate kinase and identification of a single amino acid substitution (Thr384----Met) affecting enzymatic stability in a pyruvate kinase variant (PK Tokyo) associated with hereditary hemolytic anemia.

“…The Thr3m residue in human R-PK is one of the well-conserved amino acid residues among yeast (28), rat R (4), rat L (26,27), rat M1, M2 (6), chicken M (25), cat M1 (29), and human L (12) and M2 (30) types. This residue is located at the end of the seventh a-helix of the A domain (29).…”

“…The amino acid sequences of PK have been determined in several species (25)(26)(27)(28), and in cat muscle PK the tertiary structure has been proposed by data from crystallographic studies (29). The Thr3m residue in human R-PK is one of the well-conserved amino acid residues among yeast (28), rat R (4), rat L (26,27), rat M1, M2 (6), chicken M (25), cat M1 (29), and human L (12) and M2 (30) types.…”

Human liver type pyruvate kinase: complete amino acid sequence and the expression in mammalian cells.

“…11 The expression of the cDNA in monkey COS cells (16) gave rise to a protein that has the authentic human L-type PK activity.…”

“…Several investigators have isolated PK cDNA and the chromosomal genes from nonhuman species-namely, yeast PK; chicken M1-type PK; and rat M1-, M2-, L-, and R-type PK (5,6,(8)(9)(10)(11)(12)(13)(14). In humans, abnormalities of the L (or R)-type PK are considered to be pathogenic for hereditary hemolytic anemia due to erythrocyte PK deficiency.…”

Phosphorylation of type‐L pyruvate kinase in intact hepatocytes Localisation of the phosphorylation site in response to both glucagon and the Ca2+‐linked agonist phenylephrine

“…The complete nucleotide sequence for rat liver pyruvate kinase has been determined [18,19] and the amino acid sequence of the enzyme predicted. These results indicate that there are two aspartylprolyl sites in the enzyme, and the protein should be cleaved into three fragments of 16.9, 28.5 and 13.5 kDa.…”