Role of lysine, tryptophan and calcium in the beta-elimination activity of a low-molecular-mass pectate lyase from Fusarium moniliformae.
An extracellular pectate lyase from Fusarium moniliformae was purified to homogeneity by affinity chromatography followed by gel filtration, with a yield of 76.5%. Laser desorption MS of the enzyme gave a molecular mass of 12,133.5 +/- 2.5 Da. The pectate lyase was a glycoprotein with a 5% carbohydrate content and had a pl value of 9.1. Atomic-emission spectrometry showed that Ca2+ was a part of the holoenzyme held by carboxy groups of the protein. These results support the hypothesis of a putative Ca2+ site suggested by Yodder, Keen and Jurnak [(1993) Science 260, 1503-1507] in the crystal structure of pectate lyase C of Erwinia chrysanthemi. Loss of Ca2+ was observed by treatment with EGTA or carboxy-modifying Woodward's reagent K, with subsequent loss of enzyme activity. Tryptophan fluorescence quenching showed that Ca2+ does not affect binding of substrate to enzyme. Chemical-modification and substrate-protection studies showed the presence of lysine and tryptophan at or near the active site of the pectate lyase. Chemically modified enzyme showed no major structural changes as determined by CD. Amino acid analyses of native, trinitrobenzenesulphonate (TBNS)-treated and substrate-protected TNBS-treated enzyme showed that a single essential residue of lysine is present at or near the active-site. Substrate-affinity studies showed that tryptophan could be essential for substrate binding, whereas lysine could be involved in the catalysis. Fluorescence quenching further confirmed the involvement of tryptophan in substrate binding. The reaction mechanism involving beta-elimination by this enzyme is discussed.
Rao
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MN
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Kembhavi
A A
AA
Pant
A
A
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002942:Circular Dichroism; D005670:Fusarium / Q000201:enzymology*; D008239:Lysine / Q000378:metabolism*; D008970:Molecular Weight; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*; D014461:Ultracentrifugation
D000596:Amino Acids; D014364:Tryptophan; D011133:Polysaccharide-Lyases; C023911:pectate lyase; D008239:Lysine; D002118:Calcium