Barrett J F, Dolinger D L, Schramm V L, Shockman G D
J Biol Chem. 1984 Oct 10;259(19):11818-27.
The action of purified N-acetylmuramoylhydrolase (muramidase, EC 3.2.1.17) of Streptococcus faecium ATCC 9790 on linear, uncross-linked, soluble, peptidoglycan chains produced by the same organism in the presence of benzylpenicillin was characterized as a processive exodisaccharidase. Specific labels, one [( 14C]Gal) added to the nonreducing ends of chains, and the other (3H from [3H]NaBH4) incorporated into the reducing ends of the chains, were used to establish that an enzyme molecule binds at the nonreducing terminus and sequentially hydrolyzes the glycosidic bonds, releasing disaccharide-peptide units. An enzyme molecule remains bond to a chain, and is not released at a detectable rate, until hydrolysis of that chain is complete. Reaction rates increased with the length of the polymer chain to give a maximum of 91 bonds cleaved/min/enzyme molecule for hydrolysis of a continuous polymeric substrate. The relationship between hydrolytic rate and glycan chain length is consistent with hydrolysis of bonds within the chain followed by slow release of enzyme from the distal, reducing terminus. This mechanism was experimentally confirmed by analysis of product formation during hydrolysis with stoichiometric mixtures of enzyme and soluble peptidoglycan chains. Kinetic analyses showed an apparent Km of 0.17 microM for the enzyme, independent of substrate polymer length. The dissociation constant for the initial enzyme-substrate complex was calculated to be 1.5 nM. Kinetic analyses are consistent with one catalytic site per enzyme molecule. The Kcat/Km value of 9 X 10(6) M-1 S-1 is near the limit imposed by diffusion for the initial hydrolytic events when long chains are hydrolyzed. The kinetic and physical properties of this muramidase are highly consistent with its location outside of the cellular permeability barrier and its ability to remain with and hydrolyze appropriate bonds in the cell wall in such an environment.
粪肠球菌ATCC 9790的纯化N - 乙酰胞壁酰水解酶（溶菌酶，EC 3.2.1.17）对该菌在苄青霉素存在下产生的线性、未交联、可溶性肽聚糖链的作用，被表征为一种连续性外切二糖酶。使用了两种特异性标记，一种是添加到链的非还原末端的[(14C]半乳糖，另一种是掺入链的还原末端的（来自[3H]硼氢化钠的3H），以确定酶分子在非还原末端结合，并依次水解糖苷键，释放二糖 - 肽单元。酶分子一直与一条链结合，在该链水解完成之前，不会以可检测的速率释放。反应速率随聚合物链长度增加，对于连续聚合物底物的水解，最大速率为每分钟每酶分子裂解91个键。水解速率与聚糖链长度之间的关系与链内键的水解，随后酶从远端还原末端缓慢释放相一致。通过用酶和可溶性肽聚糖链的化学计量混合物水解过程中产物形成的分析，实验证实了这一机制。动力学分析表明该酶的表观Km为0.17微摩尔，与底物聚合物长度无关。初始酶 - 底物复合物的解离常数经计算为1.5纳摩尔。动力学分析与每个酶分子有一个催化位点一致。当长链被水解时，9×10(6) M-1 S-1的Kcat/Km值接近初始水解事件扩散限制的极限。这种溶菌酶的动力学和物理性质与其位于细胞通透性屏障之外的位置，以及在这种环境下与细胞壁中适当键结合并水解的能力高度一致。
