1. Direct crystallographic observation of an acyl-enzyme intermediate in the elastase-catalyzed hydrolysis of a peptidyl ester substrate: Exploiting the "glass transition" in protein dynamics
Xiaochun Ding, Bjarne F Rasmussen, Gregory A Petsko, Dagmar Ringe Bioorg Chem. 2006 Dec;34(6):410-23. doi: 10.1016/j.bioorg.2006.10.002. Epub 2006 Nov 2.
The crystal structure of the acyl complex of porcine pancreatic elastase with its peptidyl ester substrate N-acetyl-ala-ala-ala-methyl ester (Ac(Ala)3OMe) has been determined at 2.5 A resolution. The complex was stabilized by exploiting the "glass transition" in protein dynamics that occurs at around -53 degrees C (220 K). Substrate was flowed into the crystal in a cryoprotective solvent above this temperature, and then the crystal was rapidly cooled to a temperature below the transition to trap the species that formed. The use of a flow cell makes the experiment a kinetic one and means that the species prior to the rate determining transition state has a chance to accumulate. The resulting crystal structure shows an acyl-enzyme intermediate in which the leaving group is absent and the carbonyl carbon of the C-terminal alanine residue is covalently bound to the gamma oxygen of the active site serine. The ester carbonyl shows no significant distortion from planarity, with the carbonyl oxygen forming one hydrogen bond with the oxyanion hole. The tripeptide is bound in an extended antiparallel beta-sheet with main chain residues of the enzyme. The geometry and interactions of this acyl-enzyme suggest that it represents a productive intermediate. To test this hypothesis, the same crystal was then warmed above the glass transition temperature and a second data set was collected. The resulting electron density map shows no sign of the substrate, indicating hydrolysis of the intermediate followed by product release. This experiment provides direct evidence for the importance of dynamic properties in catalysis and also provides a blueprint for the stabilization of other short-lived species for direct crystallographic observation.
2. Degradation of type IV (basement membrane) collagen by a proteinase isolated from human polymorphonuclear leukocyte granules
C L Mainardi, S N Dixit, A H Kang J Biol Chem. 1980 Jun 10;255(11):5435-41.
A serine esterase with potent proteolytic activity against native bovine lens capsule type IV collagen was isolated and purified from extract of human polymorphonuclear leukocytes (PMN). The type IV collagenolytic activity co-purified with N-t-benzyloxycarbonyl-L-alanine nitroanilidase, and was inhibited by phenylmethanesulfonyl fluoride and N-acetyl-Ala-Ala-Ala-Ala chloromethyl ketone. In addition, the purified enzyme had elastolytic activity, reacted with a specific antibody to PMN elastase, and, therefore, appeared to be identical with this enzyme. A simple, reproducible assay for the detection of type IV collagenase activity using insoluble bovine anterior lens capsule collagen was defined. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed that the enzyme released large molecular weight peptides (greater than 30,000) from the insoluble substrate. The enzyme was also active against native, pepsin-solubilized type IV collagen; five reaction products could be identified. These data suggest that PMN elastase may be involved in the degradation of basement membrane collagen in physiologic and pathologic states.
