Evan R. Kantrowitz

A Model of the Transition State in the Alkaline Phosphatase Reaction

A high resolution crystal structure ofEscherichia coli alkaline phosphatase in the presence of vanadate has been refined to 1.9 Å resolution. The vanadate ion takes on a trigonal bipyramidal geometry and is covalently bound by the active site serine nucleophile. A coordinated water molecule occupies the axial position opposite the serine nucleophile, whereas the equatorial oxygen atoms of the vanadate ion are stabilized by interactions with both Arg-166 and the zinc metal ions of the active site. This structural complex supports the in-line displacement mechanism of phosphomonoester hydrolysis by alkaline phosphatase and provides a model for the proposed transition state in the enzyme-catalyzed reaction.

The mechanism of the alkaline phosphatase reaction: insights from NMR, crystallography and site-specific mutagenesis.

The proposed double in‐line displacement mechanism of Escherichia coli alkaline phosphatase (AP) involving two‐metal ion catalysis is based on NMR spectroscopic and X‐ray crystallographic studies. This mechanism is further supported by the X‐ray crystal structures of the covalent phospho‐enzyme intermediate of the H331Q mutant AP and of the transition state complex between the wild‐type enzyme and vanadate, a transition state analog. Kinetic and structural studies on several genetically engineered versions of AP illustrate the overall importance of the active sites metal geometry, hydrogen bonding network and electrostatic potential in the catalytic mechanism.

An improved colorimetric assay for aspartate and ornithine transcarbamylases

Abstract An improved colorimetric assay for ornithine and aspartate transcarbamylase has been devised. The conventional method of L. M. Prescott and M. E. Jones (1969, Anal. Biochem.32, 408–419) for the detection of ureido compounds, has been optimized and standardized to a highly reproducible, sensitive, efficient, and inexpensive method for the assay of carbamyl aspartate or citrulline, the products of aspartate transcarbamylase and ornithine transcarbamylase, respectively.

E. coli aspartate transcarbamylase: Part II: Structure and allosteric interactions

Abstract Aspartate transcarbamylase catalyses and regulates the committed step in the pyrimidine biosynthesis pathway in E. coli . The combination of biochemical information together with the high resolution X-ray structure of this enzyme, makes possible an initial understanding of these functions on the molecular level.

E. coli aspartate transcarbamylase: Part I: Catalytic and regulatory functions

Abstract E. coli aspartate transcarbamylase controls pyrimidine biosynthesis by a combination of genetic, metabolic and allosteric mechanisms, leading to a balanced pool of pyrimidines and purines in the cell. A wealth of biochemical information now allows us to begin to understand this complex allosteric enzyme.

Characterization of a Monomeric Escherichia coli Alkaline Phosphatase Formed upon a Single Amino Acid Substitution

Alkaline phosphatase (AP) from Escherichia coli as well as APs from many other organisms exist in a dimeric quaternary structure. Each monomer contains an active site located 32 Å away from the active site in the second subunit. Indirect evidence has previously suggested that the monomeric form of AP is inactive. Molecular modeling studies indicated that destabilization of the dimeric interface should occur if Thr-59, located near the 2-fold axis of symmetry, were replaced by a sterically large and charged residue such as arginine. The T59R enzyme was constructed and characterized by sucrose-density gradient sedimentation, size-exclusion chromatography, and circular dichroism (CD) and compared with the previously constructed T59A enzyme. The T59A enzyme was found to exist as a dimer, whereas the T59R enzyme was found to exist as a monomer. The T59A, T59R, and wild-type APs exhibited almost identical secondary structures as judged by CD. The T59R monomeric AP has a melting temperature (Tm) of 43 °C, whereas the wild-type AP dimer has a Tm of 97 °C. The catalytic activity of the T59R enzyme was reduced by 104-fold, whereas the T59A enzyme exhibited an activity similar to that of the wild-type enzyme. The T59A and wild-type enzymes contained similar levels of zinc and magnesium, whereas the T59R enzyme has almost undetectable amounts of tightly bound metals. These results suggest that a significant conformational change occurs upon dimerization, which enhances thermal stability, metal binding, and catalysis.

Insights into the mechanisms of catalysis and heterotropic regulation of Escherichia coli aspartate transcarbamoylase based upon a structure of the enzyme complexed with the bisubstrate analogue N-phosphonacetyl-L-aspartate at 2.1 Å

A high‐resolution structure of Escherichia coli aspartate transcarbamoylase has been determined to 2.1 Å; resolution in the presence of the bisubstrate analog N‐phosphonacetyl‐L‐aspartate (PALA). The structure was refined to a free R‐factor of 23.4% and a working R‐factor of 20.3%. The PALA molecule is completely saturated with interactions to side chain and backbone groups in the active site, including two interactions that are contributed from the 80s loop of the adjacent catalytic chain. The charge neutralization of the bound PALA molecule (and presumably the substrates as well) induced by the electrostatic field of the highly positively charged active site is an important factor in the high binding affinity of PALA and must be important for catalysis. The higher‐resolution structure reported here departs in a number of ways from the previously determined structure at lower resolution. These modifications include alterations in the backbone conformation of the C‐terminal of the catalytic chains, the N‐ and C‐termini of the regulatory chains, and two loops of the regulatory chain. The high‐resolution of this structure has allowed a more detailed description of the binding of PALA to the active site of the enzyme and has allowed a detailed model of the tetrahedral intermediate to be constructed. This model becomes the basis of a description of the catalytic mechanism of the transcarbamoylase reaction. The R‐structural state of the enzyme‐PALA complex is an excellent representation of the form of the enzyme that occurs at the moment in the catalytic cycle when the tetrahedral intermediate is formed. Finally, improved electron density in the N‐terminal region of the regulatory chain (residues 1 to 7) has allowed tracing of the entire regulatory chain. The N‐terminal segments of the R1 and R6 chains are located in close proximity to each other and to the regulatory site. This portion of the molecule may be involved in the observed asymmetry between the regulatory binding sites as well as in the heterotropic response of the enzyme. Protein 1999;37:729–742. ©1999 Wiley‐Liss, Inc.

A library of novel allosteric inhibitors against fructose 1,6-bisphosphatase.

The identification of a proper lead compound for fructose 1,6-bisphosphatase (FBPase) is a critical step in the process of developing novel therapeutics against type-2 diabetes. Herein, we have successfully generated a library of allosteric inhibitors against FBPase as potential anti-diabetic drugs, of which, the lead compound 1b was identified through utilizing a virtual high-throughput screening (vHTS) system, which we have developed. The thiazole-based core structure was synthesized via the condensation of alpha-bromo-ketones with thioureas and substituents on the two aryl rings were varied. 4c was found to inhibit pig kidney FBPase approximately fivefold better than 1b. In addition, we have also identified 10b, a tight binding fragment, which can be use for fragment-based drug design purposes.

Structure of the wild-type TEM-1 β-lactamase at 1.55 Å and the mutant enzyme Ser70Ala at 2.1 Å suggest the mode of noncovalent catalysis for the mutant enzyme

One of the best-studied examples of a class A beta-lactamase is Escherichia coli TEM-1 beta-lactamase. In this class of enzymes, the active-site serine residue takes on the role of a nucleophile and carries out beta-lactam hydrolysis. Here, the structures of the wild-type and the S70G enzyme determined to 1.55 and 2.1 A, respectively, are presented. In contrast to the previously reported 1.8 A structure, the active site of the wild-type enzyme (1.55 A) structure does not contain sulfate and Ser70 appears to be in the deprotonated form. The X-ray crystal structure of the S70G mutant has an altered Ser130 side-chain conformation that influences the positions of water molecules in the active site. This change allows an additional water molecule to be positioned similarly to the serine hydroxyl in the wild-type enzyme. The structure of the mutant enzyme suggests that this water molecule can assume the role of an active-site nucleophile and carry out noncovalent catalysis. The drop in activity in the mutant enzyme is comparable to the drop observed in an analogous mutation of the nucleophilic serine in alkaline phosphatase, suggesting common chemical principles in the utilization of nucleophilic serine in the active site of different enzymes.

Why are mammalian alkaline phosphatases much more active than bacterial alkaline phosphatases

Mammalian alkaline phosphatases are 20‐30‐fold more active than the corresponding bacterial enzymes even though their amino acid sequences are 25–30% absolutely conserved. In the active‐site region there are two noticeable differences between the sequences of the bacterial and mammalian enzymes, in the Escherichia coli enzyme positions 153 and 328 are Asp and Lys, respectively, but in the mammalian enzymes His is observed at both of these positions. Site‐specific mutagenesis, genetic and X‐ray crystallographic data, which will be summarized here, suggest that the His substitutions at positions 153 and 328 are primarily responsible for the differences in properties between the bacterial and mammalian alkaline phosphatases.