Inactive and temperature-sensitive folding mutants generated by tryptophan substitutions in the membrane-bound d-lactate dehydrogenase of Escherichia coli.
A combination of site-specific mutagenesis and 19F nuclear magnetic resonance has been used to investigate the structural properties of D-lactate dehydrogenase, a membrane-associated enzyme of Escherichia coli. The protein (65,000 Da) has been labeled with 5-fluorotryptophan for 19F nuclear magnetic resonance studies. Tryptophan has been substituted for individual phenylalanine, tyrosine, isoleucine, and leucine residues at various positions throughout the enzyme molecule, and the fluorinated native and substituted tryptophan residues have been used as probes of the local environment. All 24 mutants thus generated are expressed in E. coli. Ten are fully active and purfiable following the usual procedure, while 14 either are inactive or produce low levels of activity. The amount of active enzyme produced from the low-yield mutants is dependent on the temperature at which synthesis is carried out, with more active enzyme produced at 18 degrees C than at 27, 35, or 42 degrees C. Cells grown at 27 degrees C and then incubated at 42 degrees C retain 90-100% of their activity. All of the expressed protein from the inactive mutants is Triton-insoluble, aggregated, and not readily purfiable; the inactive mutant protein appears to be improperly folded. Most of the expressed D-lactate dehydrogenase from the partially active mutants is also Triton-insoluble; a small fraction, however, is soluble in Triton and can be purified to yield active enzyme. All the purified enzymes from these low-yield mutants of D-lactate dehydrogenase have essentially normal VmaxS, and all but two have normal KmS. Once purified, the low-yield mutant enzymes are stable at 42 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
H T
HT
Truong
E A
EA
Pratt
G S
GS
Rule
P Y
PY
Hsue
C
C
Ho
D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D013696:Temperature; D014364:Tryptophan / Q000737:chemistry*
D014364:Tryptophan; D007770:L-Lactate Dehydrogenase