INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE BY PENTALENOLACTONE .2. IDENTIFICATION OF THE SITE OF ALKYLATION BY TETRAHYDROPENTALENOLACTONE
INHIBITION OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE BY PENTALENOLACTONE .2. IDENTIFICATION OF THE SITE OF ALKYLATION BY TETRAHYDROPENTALENOLACTONE
复制标题
DOI：
10.1021/bi00187a020
复制
发表时间：
1994-05-31
期刊：
影响因子：
2.9
通讯作者：
SOHNG, JK
中科院分区：
文献类型：
--
作者：
CANE, DE;SOHNG, JK
兔肌肉甘油醛-3-磷酸脱氢酶(GAPDH)与抗生素戊烯内酯(3)孵育，通过修饰同源四聚体酶每个亚单位中的单个半胱氨酸残基，导致对GAPDH的时间依赖的、不可逆转的抑制。用氚气体还原戊烯内酯得到[2，3，7，8-H-3(4)]四氢戊烯内酯(7)，它也表现出时间依赖性的不可逆失活GAPDH。确定了7的共价连接位置。对灭活的GAPDH进行胰酶消化，用反相高效液相色谱分离纯化产物，得到单标记肽。氨基酸序列分析得到Ile-Val-Ser-Asn-Ala-Ser-X-Thr-Thr-Asn-(...该序列与猪肌肉GAPDHIle-143至ASN-152高度保守的区域完全相同，只是活性部位Cys-149与四氢戊烯内酯共价结合。分子模拟被用来比较戊烯内酯(3)和庚二酸(4)，一个机理上相关的GAPDH的失活剂，和正常底物3-磷酸甘油醛(1)。最后，通过与模型硫醇的反应表明，戊烯内酯仅通过亲核攻击在伯碳C-10上进行环氧化物开环。
Incubation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with the antibiotic pentalenolactone (3) results in time-dependent, irreversible inhibition of GAPDH by modification of a single Cys residue in each subunit of the homotetrameric enzyme. Reduction of pentalenolactone with tritium gas gave [2,3,7,8-H-3(4)]tetrahydropentalenolactone (7), which also exhibited time-dependent, irreversible inactivation of GAPDH. The site of covalent attachment of 7 was determined. Tryptic digestion of inactivated GAPDH and purification of the resultant products by reverse-phase HPLC gave a single labeled peptide. Amino acid sequence analysis of the radioactive peptide gave Ile-Val-Ser-Asn-Ala-Ser-X-Thr-Thr-Asn-(...). This sequence is identical to the highly conserved region from Ile-143 to Asn-152 in pig muscle GAPDH, except for the active site Cys-149 to which the tetrahydropentalenolactone was covalently bound. Molecular modeling was used to compare both pentalenolactone (3) and heptelidic acid (4), a mechanistically related inactivator of GAPDH, with the normal substrate, glyceraldehyde 3-phosphate (1). Finally, pentalenolactone was shown by reaction with model thiols to undergo epoxide ring opening exclusively by nucleophilic attack at the primary carbon, C-10.