PMID- 8870663
OWN - NLM
STAT- MEDLINE
DCOM- 19961203
LR  - 20190501
IS  - 0264-6021 (Print)
IS  - 1470-8728 (Electronic)
IS  - 0264-6021 (Linking)
VI  - 319 ( Pt 1)
IP  - Pt 1
DP  - 1996 Oct 1
TI  - Role of lysine, tryptophan and calcium in the beta-elimination activity of a 
      low-molecular-mass pectate lyase from Fusarium moniliformae.
PG  - 159-64
AB  - 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.
FAU - Rao, M N
AU  - Rao MN
AD  - Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
FAU - Kembhavi, A A
AU  - Kembhavi AA
FAU - Pant, A
AU  - Pant A
LA  - eng
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PL  - England
TA  - Biochem J
JT  - The Biochemical journal
JID - 2984726R
RN  - 0 (Amino Acids)
RN  - 8DUH1N11BX (Tryptophan)
RN  - EC 4.2.2.- (Polysaccharide-Lyases)
RN  - EC 4.2.2.2 (pectate lyase)
RN  - K3Z4F929H6 (Lysine)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Amino Acids/analysis
MH  - Binding Sites
MH  - Calcium/*metabolism
MH  - Circular Dichroism
MH  - Fusarium/*enzymology
MH  - Lysine/*metabolism
MH  - Molecular Weight
MH  - Polysaccharide-Lyases/chemistry/*metabolism
MH  - Protein Conformation
MH  - Spectrometry, Fluorescence
MH  - Tryptophan/*metabolism
MH  - Ultracentrifugation
PMC - PMC1217749
EDAT- 1996/10/01 00:00
MHDA- 1996/10/01 00:01
PMCR- 1997/04/01
CRDT- 1996/10/01 00:00
PHST- 1996/10/01 00:00 [pubmed]
PHST- 1996/10/01 00:01 [medline]
PHST- 1996/10/01 00:00 [entrez]
PHST- 1997/04/01 00:00 [pmc-release]
AID - 10.1042/bj3190159 [doi]
PST - ppublish
SO  - Biochem J. 1996 Oct 1;319 ( Pt 1)(Pt 1):159-64. doi: 10.1042/bj3190159.