Coyette J, Ghuysen J M, Fontana R
Eur J Biochem. 1980 Sep;110(2):445-56. doi: 10.1111/j.1432-1033.1980.tb04886.x.
Streptococcus faecalis ATCC 9790 possesses seven membrane-bound penicillin-binding proteins. They have been characterized with respect to their apparent molecular weights, relative abundance, specificity profiles for 15 different beta-lactam antibiotics and stability under various conditions. In water and at 37 degrees C, all the native penicillin-binding proteins have half-lives longer than 20 h except protein 3b (half-life of about 600 min) and protein 4 (half-life of about 175 min). The short-lived 80 000-Mr protein 4 is spontaneously converted into a 73 000-Mr water-soluble, penicillin-binding protein 4. Similarly, the short-lived 82 000-Mr protein 3b seems to be the protein from which the 72 000-Mr water-soluble protein X spontaneously originates during incubation of the membranes. Release of both proteins 4 and X from the membrane is maximal under alkaline conditions; it is not inhibited by various protease inhibitors. After exposure to trypsin, the 43 000-Mr membrane-bound penicillin binding protein 6 (a DD-carboxypeptidase) gives to a 30 000-Mr water-soluble protein 6. Like the parent protein, protein 6 exhibits both DD-carboxypeptidase activity and penicillin-binding ability. With proteins 6 and 6, low dose levels of p-chloromercuribenzoate prevent both enzyme activity and combination with penicillin, thus strongly suggesting that a thiol group is involved in the enzyme active center. We have shown previously [Coyette et al. in Eur. J. Biochem. 88, 297--305 (1978) and 75, 231--239 (1977)] that the DD-carboxypeptidase protein 6 fragments the benzylpenicillin molecule with formation of phenylacetylglycine. Breakdown of the complex formed between [14C]benzylpenicillin and 14 000-Mr membrane-bound protein 1 is also 'enzyme-catalysed'. Most likely, however, the released product is penicilloate. With all the other penicillin-binding proteins whose molecular weights are intermediate between those of proteins 1 and 6, breakdown of the complexes formed with [14C]benzylpenicillin results from proteolysis and is not due to the release of the bound metabolite. None of the penicillin-binding proteins behaves, by itself, as a lethal target for beta-lactam antibiotic action on the living cells.
粪肠球菌ATCC 9790拥有七种膜结合青霉素结合蛋白。已对它们的表观分子量、相对丰度、对15种不同β-内酰胺抗生素的特异性谱以及在各种条件下的稳定性进行了表征。在水中和37℃时，除了蛋白3b（半衰期约600分钟）和蛋白4（半衰期约175分钟）外，所有天然青霉素结合蛋白的半衰期都超过20小时。寿命较短的80000道尔顿蛋白4会自发转化为73000道尔顿的水溶性青霉素结合蛋白4。同样，寿命较短的82000道尔顿蛋白3b似乎是在膜孵育过程中72000道尔顿水溶性蛋白X自发产生的来源蛋白。蛋白4和X从膜上的释放在碱性条件下最大；它不受各种蛋白酶抑制剂的抑制。用胰蛋白酶处理后，43000道尔顿的膜结合青霉素结合蛋白6（一种DD-羧肽酶）会产生30000道尔顿的水溶性蛋白6。与亲本蛋白一样，蛋白6同时具有DD-羧肽酶活性和青霉素结合能力。对于蛋白6和6，低剂量的对氯汞苯甲酸会同时抑制酶活性和与青霉素的结合，因此强烈表明一个巯基参与了酶的活性中心。我们之前已经表明[科耶特等人，《欧洲生物化学杂志》88卷，297 - 305页（1978年）和75卷，231 - 239页（1977年）]，DD-羧肽酶蛋白6会使苄青霉素分子断裂形成苯乙酰甘氨酸。[14C]苄青霉素与14000道尔顿膜结合蛋白1形成的复合物的分解也是“酶催化的”。然而，最有可能的是，释放的产物是青霉素酸盐。对于所有其他分子量介于蛋白1和6之间的青霉素结合蛋白，与[14C]苄青霉素形成的复合物的分解是由蛋白水解引起的，而不是由于结合代谢物的释放。没有一种青霉素结合蛋白自身表现为β-内酰胺抗生素对活细胞作用的致死靶点。
