The Basis for k*_cat Impairment in Prophospholipase A₂ from the Anion-Assisted Dimer Structure^,

Abstract

Kinetic results in this paper show that, contrary to earlier reports, pig pancreatic prophospholipase A(2) (proPLA2) does not hydrolyze monodisperse short chain phosphatidylcholine below the critical micelle concentration. ProPLA2 is active on an anionic interface, but at a rate that is decreased by more than 100-fold compared to that of PLA2, the active form. Solution studies show that both proPLA2 and PLA2 bind to an anionic interface and also bind a tetrahedral intermediate mimic at the active site. The 1.5 A resolution crystal structure of the anion-assisted dimer of proPLA2 reported in this paper is compared with the corresponding structure for PLA2 [Pan, Y. H., et al. (2001) Biochemistry 40, 609-617]. As a mimic for the forms bound to the anionic interface, these structures provide insights into the possible structural basis for the impaired chemical step of the zymogen. The proPLA2 dimer contained within one crystallographic asymmetric unit has one molecule of the inhibitor 1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol and is bridged by four coplanar sulfate anions. Relative to the structure of PLA2, the subunit contact surface in proPLA2 displays a tilted orientation, an altered mode of inhibitor binding, displacement of a mechanistically significant loop that includes Tyr69, and a critical active site water seen in PLA2 that is not seen in proPLA2. These differences are interpreted to suggest possible origins of the functional differences between the pro and active enzyme at an anionic interface. A structural origin of this difference is discussed in terms of the calcium-coordinated activated water mechanism of the esterolysis reaction. Together, a comparison of the structures of the anion-assisted dimers of PLA2 and proPLA2 not only offers an explanation of why the zymogen form is k(cat)-impaired and binds poorly even to the anionic interface but also supports a mechanism for the activated enzyme that includes a critical second-sphere assisting water bridging His48 and the calcium-coordinated catalytic water.

Structural Insight into the Activation Mechanism of Human Pancreatic Prophospholipase A2

“…The PROP (from Asp6 to Arg0) occupies the active site that was used for inhibitor binding in porcine pancreatic PLA2 (18). In porcine pancreatic PLA2 structure, there are six amino acid (aa) residues (Phe22, Gly30, His48, Asp49, Tyr52, and Phe106) interacting with the inhibitor MJ33, and these residues form the active site cavity.…”

“…A model for this i-face was postulated based on the crystal structure of dimeric porcine G1B (18). Questions still remain on the details of this i-face and the mechanism of its binding to membrane.…”

Group X Secreted Phospholipase A2 Proenzyme Is Matured by a Furin-like Proprotein Convertase and Releases Arachidonic Acid inside of Human HEK293 Cells

“…The sPLA 2 -IB proenzyme has a short N-terminal propeptide of 7 amino acids ending with arginine, suggesting cleavage by a trypsin-like activity (7). The propeptide has been shown to dramatically decrease the enzymatic activity by preventing catalytically productive interfacial binding to phospholipids (8,9). The enzyme is highly expressed in exocrine pancreas and is activated extracellularly by trypsin in the duodenum (7,10).…”

Crystal Structure of a Class XIB Phospholipase A2 (PLA2)

“…In subunit B the latter has strong density, whereas very little density is seen for the water coordinating His 61 , Asp 62 , and Cys 58 . The water at this position is interesting, because it corresponds to the proposed assisting water molecule, and its presence supports the water-assisted calcium-coordinate oxyanion mechanism of PLA 2 (15)(16)(17)(18). However, this assisting water has been assumed to be present only in the activated form of sPLA 2 s (56) and to be the result of the interfacial activation process (57).…”

“…The calcium ion assists by polarizing the scissile bond and by stabilizing the negative charge developing in the transition state during phospholipid hydrolysis (14). More recently, a second water molecule, bridging the Ca 2ϩ -coordinated catalytic water to the active site histidine, has been inferred to be involved in catalysis, the water-assisted calcium-coordinate oxyanion mechanism of PLA 2 (15)(16)(17)(18). An interesting aspect of PLA 2 catalysis is interfacial activation; i.e.…”