+ Site Statistics
References:
54,258,434
Abstracts:
29,560,870
PMIDs:
28,072,757
+ Search Articles
+ Subscribe to Site Feeds
Most Shared
PDF Full Text
+ PDF Full Text
Request PDF Full Text
+ Follow Us
Follow on Facebook
Follow on Twitter
Follow on LinkedIn
+ Translate
+ Recently Requested

The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli



The consequences of replacing histidine 356 in isocitrate lyase from Escherichia coli



Archives of Biochemistry and Biophysics 336(2): 309-315



Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of k-cat compared to those for wild-type (wt) enzyme (100) at 37 degree C, pH 7.3, are 18, 1, lt 1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. K-m values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log k-cat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pK-a and pK-b values for dissociation of an ionizable group on the enzyme substrate complex of lt 6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pK-m vs pH were different for the wt and H356K variant. Values of pK-a and pK-b (derived from log k-cat/K-m plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pK-b value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.

(PDF emailed within 0-6 h: $19.90)

Accession: 010002015

Download citation: RISBibTeXText

PMID: 8954579

DOI: 10.1006/abbi.1996.0562


Related references

Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. Journal of Biological Chemistry 263(5): 2477-2482, 1988

The importance of four histidine residues in isocitrate lyase from Escherichia coli. Journal of Bacteriology 176(3): 927-931, 1994

Identification of the histidine residue in Escherichia coli isocitrate lyase that reacts with diethylpyrocarbonate. Biochimica et Biophysica Acta 1122(2): 212-218, 1992

Role of phosphoenolpyruvate in the NADP-isocitrate dehydrogenase and isocitrate lyase reaction in Escherichia coli. Journal of Bacteriology 189(3): 1176-1178, 2006

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli. Journal of Bacteriology 162(1): 98-101, 1985

The Role Of Isocitrate Lyase In Escherichia Coli. Biochimica et Biophysica Acta 89: 383-384, 1964

Incorporation of atp into isocitrate lyase of escherichia coli. Journal of Cell Biology 107(6 PART 3): 187A, 1988

Phosphorylation of isocitrate lyase in Escherichia coli. Biochimie 71(9-10): 1065-1070, 1989

The inhibition of isocitrate lyase from escherichia coli by glyoxylate. Current Microbiology 21(5): 313-316, 1990

Serine319 and 321 are functional in isocitrate lyase from Escherichia coli. Current Microbiology 34(4): 205-211, 1997

In vitro phosphorylation of escherichia coli isocitrate lyase. Current Microbiology 15(2): 103-106, 1987

Escherichia coli isocitrate lyase properties and comparisons. Biochimica et Biophysica Acta 966(1): 30-35, 1988

Purification and characterization of isocitrate lyase from escherichia coli. Current Microbiology 14(6): 347-350, 1987

In vivo phosphorylation of isocitrate lyase from escherichia coli d 3h 3g 7. Biochemical & Biophysical Research Communications 153(2): 875-880, 1988