Effects of N-terminal deletion mutation on rabbit muscle lactate dehydrogenase.
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.
Zheng
Yanbin
Y
Department of Biological Science and Biotechnology, Tsinghua University, Beijing 100084, China.
Guo
Shuyuan
S
Guo
Zhi
Z
Wang
Xicheng
X
D000595:Amino Acid Sequence; D000818:Animals; D004795:Enzyme Stability; D045022:Lactate Dehydrogenases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D009154:Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D045022:Lactate Dehydrogenases