Partition analysis of an enzyme acting concurrently upon two substrates in a continuous multiwavelength assay

被引:13
作者
Thompson, JE [1 ]
Jordan, DB [1 ]
机构
[1] Dupont Co, Stine Haskell Res Ctr, Newark, DE 19714 USA
关键词
D O I
10.1006/abio.1997.2490
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
We describe a multiwavelength method for measuring an enzyme's discrimination of one substrate over another by continuously monitoring the reactions of the two substrates simultaneously. This method is generally applicable to ultraviolet-visible diode array or rapid-scanning spectrophotometers and the measurement requires a single incubation of enzyme with two substrates. Rates at each of the wavelengths may be fit globally by using a nonlinear least-squares fitting procedure which provides adequate statistical analysis. The specificity of trypsin for N-alpha-benzoyl-L-arginine p-nitroanilide (BRpNA) over N-t-butyloxycarbonyl-L-alanine-p-nitrophe nylester (BocApNP) was 2.1 as measured by the multiwavelength partition method and 2.3 by comparing the individual k(cat)/K-m's for the two substrates. Multiwavelength analysis was applied to two enzymes in the biosynthetic pathway for fungal melanin: seytalone dehydratase and trihydroxynaphthalene reductase from Magnaporthea grisea. The specificity of trihydroxynaphthalene reductase for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared to scytalone, a natural substrate for the enzyme, was 95. Scytalone dehydratase was eight-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyranone than it was for scytalone. Multiwavelength analysis was also used to measure an equilibrium constant of 0.040 for the reaction {dihydroorotate + oxonic acid <-> orotate + dihydrooxonic acid} catalyzed by dihydroorotate dehydrogenase. Advantages, limitations, and further applications of this steady-state method, which directly measures relative substrate specificities, are delineated. All studies described in this paper were at pH 7.0 and 25 degrees C. (C) 1998 Academic Press.
引用
收藏
页码:7 / 13
页数:7
相关论文
共 13 条
[1]  
Andersson A, 1996, PROTEINS, V24, P525, DOI 10.1002/(SICI)1097-0134(199604)24:4<525::AID-PROT14>3.0.CO
[2]  
2-N
[3]   SITE-SPECIFIC MUTAGENESIS OF DROSOPHILA ALCOHOL-DEHYDROGENASE - EVIDENCE FOR INVOLVEMENT OF TYROSINE-152 AND LYSINE-156 IN CATALYSIS [J].
CHEN, Z ;
JIANG, JC ;
LIN, ZG ;
LEE, WR ;
BAKER, ME ;
CHANG, SH .
BIOCHEMISTRY, 1993, 32 (13) :3342-3346
[4]   GENETIC-ANALYSIS OF MELANIN-DEFICIENT, NONPATHOGENIC MUTANTS OF MAGNAPORTHE-GRISEA [J].
CHUMLEY, FG ;
VALENT, B .
MOLECULAR PLANT-MICROBE INTERACTIONS, 1990, 3 (03) :135-143
[5]  
Fersht A., 1984, ENZYME STRUCTURE MEC
[6]   SPECIES VARIATION IN THE SPECIFICITY OF RIBULOSE-BIPHOSPHATE CARBOXYLASE-OXYGENASE [J].
JORDAN, DB ;
OGREN, WL .
NATURE, 1981, 291 (5815) :513-515
[7]   A SENSITIVE ASSAY PROCEDURE FOR SIMULTANEOUS DETERMINATION OF RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE AND OXYGENASE ACTIVITIES [J].
JORDAN, DB ;
OGREN, WL .
PLANT PHYSIOLOGY, 1981, 67 (02) :237-245
[8]  
KROKOW G, 1961, J BIOL CHEM, V236, P142
[9]   REGULATION OF SOYBEAN NET PHOTOSYNTHETIC CO2 FIXATION BY INTERACTION OF CO2, O2, AND RIBULOSE 1,5-DIPHOSPHATE CARBOXYLASE [J].
LAING, WA ;
OGREN, WL ;
HAGEMAN, RH .
PLANT PHYSIOLOGY, 1974, 54 (05) :678-685
[10]   PRELIMINARY CRYSTALLOGRAPHIC STUDIES ON SCYTALONE DEHYDRATASE FROM MAGNAPORTHE-GRISEA [J].
LUNDQVIST, T ;
WEBER, PC ;
HODGE, CN ;
BRASWELL, EH ;
RICE, J ;
PIERCE, J .
JOURNAL OF MOLECULAR BIOLOGY, 1993, 232 (03) :999-1002