Effects of N-terminal deletion mutation on rabbit muscle lactate dehydrogenase

• Published in: Biochemistry (Moscow) 2004-04, Vol.69 (4), p.401-406
• Main Authors: Zheng, Yanbin, Guo, Shuyuan, Guo, Zhi, Wang, Xicheng
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Animals; Dehydrogenase; E coli; Enzyme Stability; Enzymes; High temperature; Lactate Dehydrogenases - chemistry; Lactate Dehydrogenases - genetics; Lactate Dehydrogenases - metabolism; Molecular Sequence Data; Muscles - enzymology; Mutation; Protein Conformation; Protein Denaturation; Proteins; Rabbits; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Residues; Sequence Deletion; Structure-Activity Relationship
• ISSN: 0006-2979; 1608-3040
• DOI: 10.1023/B:BIRY.0000026195.31821.e1

Deletion mutants of rabbit muscle lactate dehydrogenase (LDH) were constructed using polymerase chain reaction (PCR) to study the roles of N-terminal residues. The coding sequences of the first 5 (LD5) and 10 (LD10) amino acids of the N-terminus were deleted and the gene was inserted into the prokaryotic expression vector pET21b. The mutant enzymes were expressed in E. coli BL21/DE3 and were purified. Then their characteristics and stabilities were studied. The results showed LDH was completely inactivated when the first 10 N-terminal amino acid residues were removed, but the mutant (LD10) could have partially restored activity in the presence of structure-making ions. The removal of the first 5 and 10 N-terminal amino acid residues did not affect the aggregation state of the enzyme, that is, LD5 and LD10 were still tetramers. The stabilities of recombinant wild-type LDH (RW-LD), LD5, and LD10 were compared by incubating them at low pH, elevated temperature, and high GuHCl. The results showed that the N-terminal deletion mutants were more sensitive to denaturing environments; they were easily inactivated and unfolded. Their instability increased and their ability to refold decreased with the increased number of amino acid residues removed from the N-terminus of LDH. These results confirm that the N-terminus of LDH plays a crucial role in stabilizing the structure and in maintaining the function of the enzyme.