Residues essential for catalytic activity of soybean β‐amylase
Residues essential for catalytic activity of soybean β‐amylase
复制标题
大豆 β-淀粉酶催化活性所必需的残基
DOI：
10.1111/j.1432-1033.1994.tb18777.x
复制
发表时间：
1994
期刊：
影响因子：
5.4
通讯作者：
C. Fukazawa
中科院分区：
文献类型：
--
作者：
A. Totsuka;H. Nong;H. Kadokawa;Chan;Y. Itoh;C. Fukazawa
为了确定哪些氨基酸残基对大豆β-淀粉酶的催化活性是必需的，对目前发现的位于β-淀粉酶家族高度保守区域的天冬氨酸、谷氨酰和半胱氨酸残基进行了脱氧寡核苷酸定点突变。中性氨基酸和酸性氨基酸分别取代酶第101位的天冬氨酸和第186位的谷氨酸，使酶的活性完全消失，但对底物类似物环麦芽六糖的圆二色谱或结合亲和力无明显影响。综合以上结果，上述两个氨基酸残基参与了大豆β-淀粉酶的催化活性。345位谷氨酸的替换使活性下降到非突变水平的6%以下，这意味着该残基也可能在/i-淀粉酶活性中发挥关键作用，尽管它可能不参与催化位点本身。相反，第95位的半胱氨酸残基被丝氨酸残基取代导致最适温度(从50℃降至30℃)急剧下降，这表明这种半胱氨酸残基对酶的热稳定性负责。8-淀粉酶(1，4-α-D-葡聚糖麦芽糖水解酶)通过从非还原末端释放β-异构体麦芽糖，使淀粉和糖原的a-L，4-糖苷键发生水解性反应。这种酶存在于许多植物和细菌物种中。虽然甘薯、大麦和大豆中的酶的物理化学性质已经得到了很好的研究[L]，但酶的催化机理还没有被阐明。对来自植物和细菌的β-淀粉酶的推导序列进行了比较，发现了三个高度保守的序列，它们很可能参与了/i-淀粉酶催化部位的片段组装[2-41]。在α-淀粉酶(1，4-a-~-葡聚糖葡聚糖水解酶)催化1，4-D-葡聚糖的水解酶的情况下，通过X射线结晶学[5-71]和定点突变[8，91]，在高度保守的区域中含有的天冬氨酰基和谷氨酰基残基已被鉴定为催化残基。尽管β-淀粉酶和α-淀粉酶的作用模式不同，一级序列之间也没有明显的相似性，但位于β-淀粉酶家族保守区域的所有天冬氨酸和谷氨酰残基都可以被认为是可能的催化残基。最近，2，3-环氧丙基α-D-吡喃葡萄糖苷亲和标记大豆β-淀粉酶的结果表明，Glu186参与了催化位点[lo]。此外，由于酶被硫代试剂如碘代乙酰胺、N-乙基马来酰亚胺和苯甲酸汞灭活，位于这些区域的半胱氨基残基也可能参与了催化反应。
To determine which amino acid residues are essential for the catalytic activity of soybean P-amylase, deoxyoligonucleotide site-directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in P-amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue. Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean P-amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non-mutant level, implying that this residue may also play a crucial role in /I-amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30”C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme. 8- Amylase (1 ,4-a-D-glucan maltohydrolase) hydrolyzes a-l,4-glucosidic linkage of starch and glycogen with liberation of P-anomeric maltose from the non-reducing ends. This enzyme is found in many plant and bacteria species. Although the physicochemical properties of the enzymes from sweet potato, barley, and soybean have been well investigated [l], the catalytic mechanism of the enzyme has not yet been elucidated. Comparison of deduced sequences of P-amylases originating from plant and bacteria revealed three highly conserved sequences which very likely are involved in the segment assembly of the catalytic site of /I-amylase [2-41. In the case of a-amylase (1,4-a-~-glucan glucanohydrolase) that catalyzes the hydrolysis of 1,4-a-D-glucans, the aspartyl and glutamyl residues contained in highly conserved regions have been identified as catalytic residues by X-ray crystallography [5 -71 and by site-directed mutagenesis [8, 91. Although the action pattern of P-amylase differs from that of a-amylase and there is no significant similarity between the primary sequences, all of the aspartyl and glutamyl residues located in the conserved regions of P-amylase family can be considered possible catalytic residues. Recently, the results of affinity labeling of soybean P-amylase with 2,3epoxy propyl a-D-glucopyranoside suggested that Glu186 was involved in the catalytic site [lo]. In addition, since the enzyme was inactivated by sulfhydryl reagents such as iodoacetamide, N-ethylmaleimide and mercuribenzoate [l , 111, the cysteinyl residues located in the regions may also participate in the catalytic reaction.