The serine protease inhibitors of the serpin family are an unusual group of proteins thought to have metastable native structures. Functionally, they are unique among polypeptide protease inhibitors, although their precise mechanism of action remains controversial. Conflicting results from previous studies have suggested that the stable serpin-protease complex is trapped in either a tight Michaelis-like structure, a tetrahedral intermediate, or an acyl-enzyme. In this report we show that, upon association with a target protease, the serpin reactive-center loop (RCL) is cleaved resulting in formation of an acyl-enzyme intermediate. This cleavage is coupled to rapid movement of the RCL into the body of the protein bringing the inhibitor closer to its lowest free energy state. From these data we suggest a model for serpin action in which the drive toward the lowest free energy state results in trapping of the protease-inhibitor complex as an acyl-enzyme intermediate.The serpins are a large family of proteins which includes most of the protease inhibitors found in blood, as well as other proteins with unrelated or unknown functions (1). Serpins act as "suicide inhibitors" that react only once with their cognate protease, forming an SDS-stable complex. Current models of serpin structure suggest that, while their overall fold is generally homologous among family members, the RCL, 1 sometimes referred to as the strained or stressed loop, is capable of adopting markedly different conformations relative to the rest of the protein structure (2-5). This conformational flexibility appears to be necessary for function but can also lead to inactivation when the loop inserts into the main body of the inhibitor, becoming the central strand of the major serpin structural motif, ␤-sheet A (6 -8). This inactive conformation was first observed in RCL-cleaved ␣ 1 -antitrypsin (␣ 1 AT) (9) and more recently in the related structure of latent plasminogen activator inhibitor 1 (PAI-1) (10). Loop insertion leads to a large increase in thermal stability, presumably due to reorganization of the five-stranded ␤-sheet A from a mixed parallel-antiparallel arrangement to a six-stranded, predominantly antiparallel, ␤-sheet (11-14). This dramatic stabilization has led to the suggestion that native inhibitory serpins may be metastable structures, kinetically trapped in a state of higher free energy than their most stable thermodynamic state. Such an energetically unfavorable structure would almost certainly be subject to negative selection, and thus its retention in all inhibitory serpins implies that it has been conserved for functional reasons. Currently, the role of loop mobility in serpin function and the structure of the serpin-protease complex are controversial (15)(16)(17)(18)(19)(20)(21)28). In the late 1970s, it was reported that serpins were unlike other tight binding protease inhibitors and formed covalent ester linkages with enzymes (15). However, these conclusions were based on SDS-PAGE analysis of denatured complexes leaving the na...

Human aquaporin 5 (HsAQP5) facilitates the transport of water across plasma membranes and has been identified within cells of the stomach, duodenum, pancreas, airways, lungs, salivary glands, sweat glands, eyes, lacrimal glands, and the inner ear. AQP5, like AQP2, is subject to posttranslational regulation by phosphorylation, at which point it is trafficked between intracellular storage compartments and the plasma membrane. Details concerning the molecular mechanism of membrane trafficking are unknown. Here we report the x-ray structure of HsAQP5 to 2.0-Å resolution and highlight structural similarities and differences relative to other eukaryotic aquaporins. A lipid occludes the putative central pore, preventing the passage of gas or ions through the center of the tetramer. Multiple consensus phosphorylation sites are observed in the structure and their potential regulatory role is discussed. We postulate that a change in the conformation of the C terminus may arise from the phosphorylation of AQP5 and thereby signal trafficking.membrane protein ͉ trafficking ͉ crystallography ͉ water channel protein ͉ heterologous overexpression A quaporins (1) facilitate the flow of water across cellular membranes while preserving ion concentration gradients. By maintaining water homeostasis within cells, aquaporin family members play essential physiological roles within all kingdoms of life. They form a large superfamily containing both pure water channels, and channels also permeable to other small polar molecules such as glycerol (2). Thirteen human aquaporin (AQP) isoforms have been identified, and they govern a broad spectrum of physiological functions (2, 3). Examples include concentration of urine in the kidneys, release of tears and maintenance of lens transparency in the eye, maintenance of water homeostasis within the brain, the extrusion of sweat from the skin, control of glycerol concentration in fat metabolism, and facilitation of cell migration during angiogenesis.X-ray and electron diffraction studies have yielded crystal structures of mammalian AQP0 (4-6), AQP1 (7, 8), AQP2 (9), and AQP4 (10), plant SoPIP2;1 (11, 12), bacterial AQPZ (13,14), and GlpF (15), and the archaeal AQPM (16). These structures establish that phylogenetically and functionally diverse AQPs arrange as homotetramers, each protomer containing six highly conserved transmembrane (TM) ␣-helices. Two half-helices form a pseudoseventh TM helix because of the insertion of loops B and E into the membrane from opposite sides, placing both copies of the highly conserved Asn-Pro-Ala (NPA) AQP signature motif near the center of the water channel. The conserved aromatic/arginine (ar/R) constriction region imposes substrate selectivity on the channel (17). A consensus has emerged from molecular dynamics simulations regarding the mechanism of water transport and ion exclusion (18) establishing that an electrostatic potential barrier peaking at the NPA region prevents the cotranslocation of protons through the channel.Although the tissue-specific expressio...

The enzyme dUTPase catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate, thereby preventing a deleterious incorporation of uracil into DNA. The best known dUTPase is that from Escherichia coli, which, like the human enzyme, consists of three identical subunits. In the present work, the catalytic properties of the E. coli dUTPase were investigated in the pH range 5-11. The enzyme was found to be highly specific for dUTP and discriminated both base and sugar as well as the phosphate moiety (bound dUDP was not hydrolyzed). The second best substrate among the nucleotides serving as building blocks for DNA was dCTP, which was hydrolyzed an astonishing 10 5 times less efficiently than dUTP, a decline largely accounted for by a higher K m for dCTP. With dUTP⅐Mg as substrate, k cat was found to vary little with pH and to range from 6 to 9 s ؊1. K m passed through a broad minimum in the neutral pH range with values approaching 10 ؊7 M. It increased with deprotonation of the uracil moiety of dUTP and showed dependence on two ionizations in the enzyme, exhibiting pK a values of 5.8 and 10.3. When excess dUTPase was reacted with dUTP⅐Mg at pH 8, the two protons transferred to the reaction medium were released in a concerted mode after the rate-limiting step. The Mg 2؉ ion enhances binding to dUTPase of dUTP by a factor of 100 and dUDP by a factor of 10. Only one enantiomer of the substrate analog 2-deoxyuridine-5-(␣-thio)-triphosphate was hydrolyzed by the enzyme. These results are interpreted to favor a catalytic mechanism involving magnesium binding to the ␣-phosphate, rate-limiting hydrolysis by a shielded and activated water molecule and a fast ordered desorption of the products. The results are discussed with reference to recent data on the structure of the E. coli dUTPase⅐dUDP complex.The enzyme dUTPase (EC 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is an important factor for the prevention of uracil incorporation into DNA (1). Along with the reduced nucleotides required for the synthesis and repair of DNA, the ribonucleotide reductase converts UDP to dUDP. The latter becomes phosphorylated to dUTP, which is readily mistaken for dTTP by DNA polymerase and, hence, may become incorporated into DNA (2). The absence of dUTPase triggers an elevated recombination frequency and an abnormal mutation rate and leads to the accumulation of short DNA fragments as intermediates in the DNA metabolism (3). The adverse effects of a low dUTPase activity suggest that inhibitors of this enzyme may find a role as cytostatic drugs in cancer therapy (4). dUTPase is widespread in nature and has been found in a variety of eukaryotic and prokaryotic organisms as well as in many viruses (5-8). It has been proven to be essential for the viability of Escherichia coli (9) and Saccharomyces cerevisiae (10). In some organisms, a second major role for dUTPase is to provide dUMP for the synthesis of dTTP (11).The three-dimensional structure of dUTPase from E. coli has been determined to 1.9-Å resolution (12) and re...

A mutant recombinant plasminogen activator inhibitor 1 (PAI-1) was created (Ser-338-->Cys) in which cysteine was placed at the P9 position of the reactive center loop. Labeling this mutant with N,N'-dimethyl-N-(acetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene diamine (NBD) provided a molecule with a fluorescent probe at that position. The NBD-labeled mutant was almost as reactive as wild type but was considerably more stable. Complex formation with tissue or urokinase type plasminogen activator (tPA or uPA), and cleavage between P3 and P4 with a catalytic concentration of elastase, all resulted in identical 13-nm blue shifts of the peak fluorescence emission wavelength and 6.2-fold fluorescence enhancements. Formation of latent PAI showed the same 13-nm spectral shift with a 6.7-fold fluorescence emission increase, indicating that the NBD probe is in a slightly more hydrophobic milieu. These changes can be attributed to insertion of the reactive center loop into the beta sheet A of the inhibitor in a manner that exposes the NBD probe to a more hydrophobic milieu. The rate of loop insertion due to tPA complexation was followed using stopped flow fluorimetry. This rate showed a hyperbolic dependence on tPA concentration, with a half-saturation concentration of 0.96 microM and a maximum rate constant of 3.4 s-1. These results demonstrate experimentally that complexation with proteases is presumably associated with loop insertion. The identical fluorescence changes obtained with tPa.PAI-1 and uPA.PAI-1 complexes and elastase-cleaved PAI-1 strongly suggest that in the stable protease-PAI-1 complex the reactive center loop is cleaved and inserted into beta sheet A and that this process is central to the inhibition mechanism.

Michaelis complex, acylation, and conformational change steps were resolved in the reactions of the serpin, plasminogen activator inhibitor-1 (PAI-1), with tissue plasminogen activator (tPA) and trypsin by comparing the reactions of active and Ser 195-inactivated enzymes with site-specific fluorescent-labeled PAI-1 derivatives that report these events. Anhydrotrypsin or S195A tPA-induced fluorescence changes in P1'-Cys and P9-Cys PAI-1 variants labeled with the fluorophore, NBD, indicative of a substrate-like interaction of the serpin reactive loop with the proteinase active-site, with the P1' label but not the P9 label perturbing the interactions by 10-60-fold. Rapid kinetic analyses of the labeled PAI-1-inactive enzyme interactions were consistent with a single-step reversible binding process involving no conformational change. Blocking of PAI-1 reactive loop-beta-sheet A interactions through mutation of the P14 Thr --> Arg or annealing a reactive center loop peptide into sheet A did not weaken the binding of the inactive enzymes, suggesting that loop-sheet interactions were unlikely to be induced by the binding. Only active trypsin and tPA induced the characteristic fluorescence changes in the labeled PAI-1 variants previously shown to report acylation and reactive loop-sheet A interactions during the PAI-1-proteinase reaction. Rapid kinetic analyses showed saturation of the reaction rate constant and, in the case of the P1'-labeled PAI-1 reaction, biphasic changes in fluorescence indicative of an intermediate resembling the noncovalent complex on the path to the covalent complex. Indistinguishable K(M) and k(lim) values of approximately 20 microM and 80-90 s(-1) for reaction of the two labeled PAI-1s with trypsin suggested that a diffusion-limited association of PAI-1 and trypsin and rate-limiting acylation step, insensitive to the effects of labeling, controlled covalent complex formation. By contrast, differing values of K(M) of 1.7 and 0.1 microM and of k(lim) of 17 and 2.6 s(-1) for tPA reactions with P1' and P9-labeled PAI-1s, respectively, suggested that tPA-PAI-1 exosite interactions, sensitive to the effects of labeling, promoted a rapid association of PAI-1 and tPA and reversible formation of an acyl-enzyme complex but impeded a rate-limiting burial of the reactive loop leading to trapping of the acyl-enzyme complex. Together, the results suggest a kinetic pathway for formation of the covalent complex between PAI-1 and proteinases involving the initial formation of a Michaelis-type noncovalent complex without significant conformational change, followed by reversible acylation and irreversible reactive loop conformational change steps that trap the proteinase in a covalent complex.