复合淀粉酶酶解生淀粉机理探讨

以生土豆淀粉为原料,考察了复合生淀粉酶水解机制。发现单个糖化酶的酶解遵循Michaelis-Menten机制,而α-淀粉酶的酶解不遵循Michaelis-Menten机制;水解过程中复合酶酶解产物d[G]／dt的变化说明α-淀粉酶能很好地协同糖化酶水解生淀粉,其效果不仅仅是两者的简单相加。     

生淀粉糖化酶催化位点氨基酸及酶合成调控的初步研究

通过对Rhizopus OR-1UVN菌种所产生淀粉糖化酶在不同底物不同缓冲溶液条件下酶最适pH的测定,推测出该生淀粉糖化酶活力中心催化位点氨基酸是天冬氨酸(Asp)和谷氨酸(Glu)。实验证明5～50mg/mL浓度葡萄糖对生淀粉糖化酶没有抑制作用。分别以浓度<5mg/mL葡萄糖和淀粉为碳源的培养基进行不同碳源发酵实验,发现以淀粉为碳源的培养基Ⅰ发酵15h开始产生淀粉糖化酶,以葡萄糖为碳源的培养基Ⅱ发酵35h开始产酶(葡萄糖浓度<8mg/mL),而且前者菌体较后者少,由此可知葡萄糖对产酶有阻遏作用。实验还发现解阻遏熟淀粉糖化酶的葡萄糖浓度(15mg/mL)比生淀粉糖化酶的要高。由于葡萄糖的阻遏作用不发生在翻译水平,而发生在转录水平上,而且生淀粉糖化酶(G1)与熟淀粉糖化酶(G2)来自同一条DNA链,可以推测存在mRNA的拼接。通过以生淀粉为碳源的比较实验,发现生淀粉对生淀粉糖化酶形成的诱导作用可能主要是通过mRNA拼接的调节来实现的。     

双酶法水解辐照改性马铃薯淀粉动力学研究

为了解辐照改性马铃薯淀粉的酶解特性,用α-淀粉酶和糖化酶同时作用于马铃薯原淀粉和经400 kGy剂量辐照处理后淀粉,考察了pH值、酶解温度、α-淀粉酶用量、糖化酶用量对反应速率的影响.以米氏方程为基础,用Lineweaver-Burk法求解动力学参数.结果表明,辐照后马铃薯淀粉的酶解反应速率明显高于马铃薯原淀粉.在单一水解体系中,α-淀粉酶和糖化酶对辐照前后马铃薯淀粉的降解都遵循Michaelis-Menten方程,α-淀粉酶的Km分别为11.343 mg· mL-1和9.386 mg· mL-1,Vmax分别为0.406 mg（mL·min）-1和1.079 mg（mL·min）-1;糖化酶的Km分别为10.307 mg· mL-1和8.905 mg·mL-1,Vmax分别为0.338 mg（mL·min）-1和0.821mg（mL·min）-1;水解产物葡萄糖对反应体系具有竞争性抑制剂的作用,其抑制常数Ki分别为1.298 mg·mL-1和0.934 mg·mL-1.研究结果表明辐照有效提高了马铃薯淀粉的酶解反应活性.     

低温生淀粉糖化酶菌株RS01分离及其酶学性质

自渤海湾海泥中分离得到一株产低温生淀粉糖化酶能力较强的菌株RS01,经形态学、生理生化特性及16S rRNA分析将其鉴定为气单胞菌属。对该菌株产的低温生淀粉糖化酶酶学性质进行初步研究,结果表明其最适酶反应温度为30℃,酶的热稳定性比较差,最适pH为5.4,经TLC鉴定酶解产物中有葡萄糖,表明该分离菌株具有产低温生淀粉糖化酶的能力。     

Aspergillus sp. RSD生淀粉糖化酶的分离纯化及酶学性质

【目的】纯化得到一种生淀粉糖化酶,并对其酶学性质进行分析。【方法】从曲霉RSD发酵液中,经过硫酸铵分级盐析,HiPrep DEAE FF16/10弱阴离子交换层析,凝胶过滤层析,Hiprep 16/10 source 30S阳离子交换层析最终纯化出一种电泳纯的生淀粉酶。【结果】粗酶液纯化倍数为12.65倍,活力回收率为9.02%,SDS-PAGE结果显示该酶的相对分子质量约为82 kD。对该酶的酶学性质分析结果表明,该酶最适作用温度为50°C,在50°C以下稳定性很好,对高温较为敏感;最适作用pH为4.5,在pH 3.5-7.0范围内酶活力较为稳定,在40°C、pH 4.6条件下以可溶性淀粉为底物时的Km值和Vmax值分别为7.44 g/L和1.45 g/(L·min);金属离子对酶活性的影响试验表明,Fe2+对该酶具有显著激活效果,EDTA、Cu2+、K+对该酶酶活力有不同程度的抑制作用;底物特异性研究表明该酶对麦芽糊精具有较高酶活力。【结论】与市售糖化酶及生淀粉糖化酶相比,该酶对生淀粉的降解能力更高,在工业应用上有较好的前景。     

两步法硫酸水解热凝胶生产β-1,3-葡寡糖

对硫酸水解热凝胶制备β-1,3-葡寡糖进行了研究。首先建立了葡寡糖的分析方法,采用半制备HPLC分离热凝胶水解液得到β-1,3-葡寡糖,利用ESI-MS和TLC技术对分离组分进行了成分鉴定。考察了反应温度和反应时间对热凝胶寡糖收率的影响,结果表明,两步法硫酸水解获取低聚合度β-1,3-热凝胶寡糖(DP 2~6)的较优水解条件为:1.0 mol/L硫酸于70℃水解1%热凝胶6h,回收水解残留物后于80℃继续水解3 h;对于较高聚合度寡糖(DP 7~10)两步水解时间分别为4 h和1 h较为合适。     

大曲来源淀粉利用型乳酸菌的筛选及其淀粉利用特性

【背景】乳酸菌是面包、馒头等发酵食品中的重要功能微生物,对改善质地和风味均具有重要作用。淀粉利用能力高的乳酸菌,因其能够在生面粉中更好地定殖而具有重要的应用价值。【目的】筛选获得淀粉水解型乳酸菌并研究其淀粉利用特性。【方法】以浓香型白酒大曲为筛选源,采用淀粉基质碳源对大曲中乳酸菌进行定向富集,结合淀粉发酵能力筛选高淀粉利用能力菌株,并对筛选得到的优良菌株展开淀粉酶表达及其酶活力研究。【结果】以贮存3-6个月的大曲为优秀筛选源,以生面糊传代富集方法可较快筛选出具有良好淀粉利用能力的乳杆菌,主要物种为植物乳杆菌和类食品乳杆菌。对其中一株具有淀粉利用能力的类食品乳杆菌LBM12001的淀粉水解特征和淀粉酶活力展开研究,该菌株淀粉水解能力达10 g/L,并且其在面糊中具有良好的定殖能力;酶活力测定表明,其α-淀粉酶和麦芽糖淀粉酶为胞外酶;麦芽糖淀粉酶水解淀粉的最适pH值为3.5,比酶活为1 240 U/mg。【结论】建立起从我国传统白酒发酵大曲中高效筛选淀粉水解型乳酸菌的富集筛选方法,以及菌株的水解能力评价方法,获得的胞外麦芽糖淀粉酶分泌型乳杆菌在酸面团、馒头等需进行生面粉发酵食品的生产中具有重要应用前景。     

新茁霉多糖酶和淀粉茁霉多糖酶

新茁霉多糖酶水解茁霉多糖的α-1,4糖苷键产生潘糖,淀粉茁霉多糖酶水解茁霉多糖的α-1,6糖苷键产生麦芽三糖,二者又都能水解淀粉的α-1,4和α-1,6糖苷键。这两类酶都属于α-淀粉酶家族。从嗜热菌和高温厌氧菌中分离的这两类新酶种一般热稳定性都很好,是生产寡糖和在淀粉高温液化和糖化中很有发展前途的酶种。本文列表比较了各种新茁霉多糖酶和淀粉茁霉多糖酶的性质,并对这些酶蛋白的氨基酸顺序和淀粉酶共有序列进行了分析比较。     

嗜热菌来源的生淀粉酶分离纯化及其酶学性质

从嗜热菌库中分离到两株能水解生淀粉的菌株173和174,通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明,所分离两株菌属于Geobacillus属的细菌.液体摇瓶发酵菌株173、174,其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL.通过生淀粉吸附-熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化,得到纯化的RSDE173和RSDE174,纯化倍数分别为50和29,活力回收率分别为34%和41%.有关RSDE173和RSDE174酶学性质研究显示.对熟淀粉水解的最适作用温度均为70℃,而对生淀粉水解则分别在50℃～60℃和40℃～60℃下表现出高水解活力;对不同底物的最适作用pH值均为5.0～5.5;它们对大多数试验离子的敏感性较低,但个别离子如Co2 、Cu'2 对RSDE173或u'2 对RSDE174的酶活力有一定的抑制作用.纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同.RSDE173底物专一性顺序为红薯淀粉>小麦淀粉>玉米淀粉>木薯淀粉>糯米淀粉;而RSDE174的糯米淀粉>小麦淀粉>红薯淀粉>玉米淀粉>木薯淀粉.RSDE173对生红薯淀粉有很好的降解,其水解糊化淀粉与生红薯淀粉的比值为1.48;而RSDE174优先降解生糯米淀粉,其相应比值为1.69.     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

Similarity of kinetics of three types of myeloperoxidase from human leukocytes and four types from HL-60 cells

Km values for H2O2 and Vmax values for three types of myeloperoxidase (MPO) from human leukocytes (MPO-I, -II, and -III) and four types from human myeloid leukemia HL-60 cells (MPO-IA, -IB, -II, and -III) were determined. Km values of human leukocyte MPOs decreased with increasing pH from 4.4 to 6.2 and increased with increasing NaCl concentration from 0.025 to 0.14 M. There was no significant difference among Km values of leukocyte MPO-I, -II, and -III. NaBr also showed a tendency similar to that of NaCl with regard to the effects of pH and halide concentration on Km values. However, Km values in the presence of NaBr were lower than those in the presence of NaCl. Effects of pH and NaCl concentration on Vmax values of MPO-I, -II, and -III were also examined. Vmax values of MPO-I, -II, and -III were higher at pH 4.9 and 5.4 and increased with increasing NaCl concentration. In addition, no difference was observed between Km values of leukocyte and those of HL-60 cells. MPO-IB, the half-molecular-weight enzyme of HL-60 cells, also had the same Km values as the others. Furthermore, inhibition of the activities of seven MPOs of leukocytes and HL-60 cells by H2O2 was similarly observed at concentrations above 1 mM at pH 5.4 but not at pH 4.4. These results indicate that there is no difference in the affinity to H2O2 among leukocyte MPO-I, -II, and -III and HL-60 cell MPO-IA, -IB, -II, and -III.     

C-Terminal region of human NAD+-dependent 15-hydroxyprostaglandin dehydrogenase is involved in the interaction with prostaglandin substrates.

NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) catalyzes the oxidation of the 15(S) hydroxyl group of prostaglandins to a 15-keto group resulting in a significant reduction of the biological activities of prostaglandins. Although the key residues involved in NAD+ binding and in catalytic activity have been partially identified, the sites of interaction of the enzyme with the prostaglandin substrates are yet to be determined. Homology analysis of the primary structures of 15-PGDH from human, mouse and rat indicates that the sequences are almost homologous except for two regions near the C-terminus. The involvement of the C-terminal region in catalytic activity was examined by studies on C-terminally truncated enzymes and on human/rat chimeric enzymes. When three to four amino acids were removed successively from the C-terminal end of human 15-PGDH, the truncated enzymes exhibited decreasing Vmax/Km ratios and increasing Km values for PGE2 as the chain was shortened. Similarly, when the C-terminal 14 amino acids of human 15-PGDH were replaced by the C-terminal 14 amino acids of rat 15-PGDH or vice versa, the Vmax/Km ratios and the Km values for prostaglandin E2 of the chimeric enzymes were in between those of the two wild-type enzymes. This indicates that the catalytic effectiveness of human 15-PGDH decreases as the C-terminal region is gradually removed or replaced by rat sequences. The C-terminal region appears to be more important for the interaction of the enzyme with the prostaglandin substrates than with the coenzyme.     

Differences in substrate specificities of monoamine oxidase A from human liver and placenta.

The substrate specificities of monoamine oxidase (MAO) A isolated from human placenta and of human liver expressed in yeast have been compared in homogeneous preparations with respect to Vmax and Km values for natural and synthetic substrates and Ki values for competitive inhibitors. MAO A from these two sources is known to differ in at least 5 amino acid residues. While the Km and Ki values were found to be nearly identical in the enzymes from these two sources, the Vmax differed significantly on bulky synthetic substrates.     

The effect of pH, temperature, and D2O on the activity of porcine synovial collagenase and gelatinase

The pH dependence of Vmax and Vmax/Km for hydrolysis of Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by porcine synovial collagenase and gelatinase was determined in the pH range 5-10. Both enzymes exhibited bell-shaped dependencies on pH for these two kinetic parameters, indicating that activity is dependent on at least two ionizable groups, one of which must be unprotonated and the other protonated. For collagenase, Vmax/Km data indicate that in the substrate-free enzyme, these groups have apparent pK values of 7.0 and 9.5, while the Vmax profile indicates similar pK values of 6.8 and 10.1 for the enzyme-substrate complex. The corresponding pH profiles of gelatinase were similar to those of collagenase, indicating the importance of groups with apparent pK values of 5.9 and 10.0 for the free enzyme and 5.9 and 11.1 for the enzyme-substrate complex. When these kinetic constants were determined in D2O using the peptide substrate, there was no significant effect on Vmax or Km for collagenase or Km for gelatinase. However, there was a deuterium isotope effect of approximately 1.5 on Vmax for gelatinase. These results indicate that a proton transfer step is not involved in the rate-limiting step for collagenase, but may be limiting with gelatinase. The Arrhenius activation energies for peptide bond hydrolysis of the synthetic peptide as well as the natural substrates were also determined for both enzymes. The activation energy (81 kcal) for hydrolysis of collagen by collagenase was nine times greater than that determined for the synthetic substrate (9.2 kcal). In contrast, the activation energy for hydrolysis of gelatin by gelatinase (26.3 kcal) was only 2.4 times greater than that for the synthetic substrate (11 kcal).     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Phospholipases. III. Effects of ionic surfactants on the phospholipase-catalyzed hydrolysis of unsonicated egg lecithin liposomes.

Apparent values of Km and Vmax have been measured for catalysis of hydrolysis of unsonicated egg lecithin liposomes, activated through addition of 0.4 M n-hexanol, by phospholipases A2 from bee and snake venoms and by phospholipase C from Clostridium welchii as a function of the concentration of three surfactants: hexadecylamine, hexadecyltrimethylammonium bromide, and dihexadecyl phosphate. For all three enzymes, values of Km and Vmax show little or no dependence on the concentration of these ionic surfactants, demonstrating that the liposomal surface charge is not a crucial factor in determining susceptibility to phospholipase-catalyzed hydrolysis.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Site-directed mutagenesis of Pro-17 located in the glycine-rich region of adenylate kinase

Proline 17 in the glycine-rich region of adenylate kinase was replaced by Gly (the Gly-mutant) or Val (the Val-mutant) by site-directed mutagenesis. The mutant enzymes were purified to homogeneous states on sodium dodecyl sulfate-gel electrophoresis after solubilization of the proteins from the pellets of cell lysates of Escherichia coli. The apparent Km values of the Gly- and the Val-mutants for AMP increased approximately 7- and 24-fold, respectively, as compared with that of the wild-type enzyme. The apparent Km values for ATP also increased 7- and 42-fold in the Gly- and Val-mutants, respectively. In contrast, Vmax values of both mutant enzymes were comparable to that of the wild-type enzyme. These results suggest that Pro-17 plays an important role for the binding of substrates, but not for catalytic efficiency, although it does not directly interact with substrates. Adenosine diphosphopyridoxal, which specifically modifies Lys-21 in adenylate kinase (Tagaya, M., Yagami, T., and Fukui, T. (1987) J. Biol. Chem. 262, 8257-8261), inactivated the wild-type and mutant enzymes at almost the same rates. Interestingly, both mutant enzymes showed higher specificities for adenine nucleotides than the wild-type enzyme. Both mutant enzymes were less resistant than the wild-type enzyme against inactivation at elevated temperatures or by treatment with trypsin. It would appear that most of the properties of the mutant enzymes may be explained on the basis of a need for conformational flexibility of the loop which includes Pro-17 for substrate binding.     

Structural function of residue-209 in coumarin 7-hydroxylase (P450coh). Enzyme-kinetic studies and site-directed mutagenesis

Residue-209 plays a critical role in determining the substrate and product specificity of cytochrome P450coh. In order to investigate further the structural function of residue-209 in coumarin 7-hydroxylase reaction, we measured the enzyme-kinetic properties of wild-type P450coh and its mutants in which residue-209 was substituted with various amino acids. In general, the Km and Vmax values for coumarin increased as the size of residue-209 became smaller and Vmax values decreased. The size of residue-209, therefore, was a principle factor determining Km, Kd, and Vmax values of P450coh. Although the polarity and charge also increased the Km value consistently, they altered Vmax and Kd values in an irregular manner. The substitution of serine for residue-209 increased the Vmax, while the substitution of lysine decreased it. Coumarin 7-hydroxylase activity was inhibited weakly by indan, but competitively and strongly by 2-coumaranone. Moreover, Ki values for the inhibitor were similar to Km values of the corresponding, mutated P450s. The results indicate, therefore, that residue-209 is localized in a proposed substrate-binding sequence 1 which binds to the 2-keto group of coumarin and directs its 7-position toward the sixth ligand of heme. Consequently, the identity of residue-209 determines not only the binding of coumarin in P450coh, but also the other reaction step(s) of coumarin 7-hydroxylation.