A novel intermediate in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase.
An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.
Acharya
J K
JK
Department of Biochemistry, Indian Institute of Science, Bangalore.
Rao
N A
NA
D000818:Animals; D002942:Circular Dichroism; D012696:Glycine Hydroxymethyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D012663:Semicarbazides / Q000737:chemistry*; D012756:Sheep; D013057:Spectrum Analysis
D012663:Semicarbazides; C005151:thiosemicarbazide; D012696:Glycine Hydroxymethyltransferase