A chalcone isomerase was purified to apparent homogeneity from develop
ing fruit tissue of Citrus sinensis by gel filtration, hydrophobic int
eraction chromatography and anion exchange chromatography. A single pr
otein band was observed on SDS-PAGE gels, corresponding to a M(r) of 2
7 800. In addition, the M(r) of the native protein was determined as 2
7 200 using analytical gel filtration. The enzyme exhibited micro-hete
rogeneity on native PAGE gels. Enzyme staining indicated the presence
of four activity bands. Four protein bands were also observed on isoel
ectric focusing gels with pI values of 6.55, 6.38, 6.35 and 6.04. Char
ge-heterogeneity could not be ascribed to glycosylation as the enzyme
was not found to be a glycoprotein. The enzyme catalysed isomerization
was found to be optimal at pH 7.4; and an energy of activation of 31.
9 kJ mol(-1) was calculated. The enzyme cyclized only naringenin chalc
one of all the aglycone substrates tested and a K-m of 4 mu M and V-ma
x of 67 mkat kg(-1) were determined for this substrate. No activity wa
s observed with chalcone glycosides. The turnover number was 1.87 sec(
-1) and the catalytic efficiency 4.62 x 10(5) M(-1)sec(-1). Competitiv
e inhibition of enzyme activity was observed with naringenin (K-i = 18
0 mu M), quercetin (K-i =45 mu M) and morin (K-i = 30 mu M).