Substituent Effects on Carbon Acidity in Aqueous Solution and at Enzyme Active Sites

Methods are described for the determination of pK as for weak carbon acids in water. The application of these methods to the determination of the pK as for a variety of carbon acids including nitriles, imidazolium cations, amino acids, peptides and their derivatives and, α-iminium cations is presented. The substituent effects on the acidity of these different classes of carbon acids are discussed, and the relevance of these results to catalysis of the deprotonation of amino acids by enzymes and by pyridoxal 5′-phosphate is reviewed. The procedure for estimating the pK a of uridine 5′-phosphate for C-6 deprotonation at the active site of orotidine 5′-phosphate decarboxylase is described, and the effect of a 5-F substituent on carbon acidity of the enzyme-bound substrate is discussed.1 Introduction2 The Carbon Acidity of Ethyl Thioacetate3 The Carbon Acidity of Carboxylic Acid Derivatives4 The Carbon Acidity of Imidazolium Cations5 The α-Carbon Acidity of Amino Acids, Peptides and Their Derivatives6 Electrophilic Catalysis of Deprotonation of Amino Acids: The α-Carbon Acidity of Iminium Cations7 pK as for Carbon Acids at Enzyme Active Sites8 Concluding Remarks

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Nonlinear Vibrations in a 2-Dimensional Protein Cluster Model with Linear Bonds

Zeitschrift für Physikalische Chemie ◽

10.1524/zpch.2000.214.1.065 ◽

2000 ◽

Vol 214 (1) ◽

Cited By ~ 9

Author(s):

E.G. Shidlovskaya ◽

L. Schimansky-Geier ◽

Yu.M. Romanovsky

Keyword(s):

Active Site ◽

Internal Resonance ◽

Cluster Model ◽

Active Sites ◽

Nonlinear Vibrations ◽

Nonlinear Phenomena ◽

Two Dimensional ◽

Dimensional Model ◽

Two Dimensional Model

A two dimensional model for the substrate inside a pocket of an active site of an enzyme is presented and investigated as a vibrational system. The parameters of the system are evaluated for α-chymotrypsin. In the case of internal resonance it is analytically and numerically shown that the energy concentrated on a certain degree of freedom might be several times larger than in the non-resonant case. Additionally, the system is driven by harmonic excitations and again energy due to nonlinear phenomena is redistributed inhomogeneously. These results may be of importance for the determination of the rates of catalytic events of substrates bound in pockets of active sites.

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On the possible origin of protein homochirality, structure, and biochemical function

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1908241116 ◽

2019 ◽

Vol 116 (52) ◽

pp. 26571-26579 ◽

Cited By ~ 3

Author(s):

Jeffrey Skolnick ◽

Hongyi Zhou ◽

Mu Gao

Keyword(s):

Amino Acids ◽

Active Site ◽

Ribosomal Proteins ◽

Active Sites ◽

Cell Formation ◽

Binding Pocket ◽

Nucleotide Biosynthesis ◽

Native Proteins ◽

Structural And Functional Properties ◽

Biochemical Function

Living systems have chiral molecules, e.g., native proteins that almost entirely contain L-amino acids. How protein homochirality emerged from a background of equal numbers of L and D amino acids is among many questions about life’s origin. The origin of homochirality and its implications are explored in computer simulations examining the stability and structural and functional properties of an artificial library of compact proteins containing 1:1 (termed demi-chiral), 3:1, and 1:3 ratios of D:L and purely L or D amino acids generated without functional selection. Demi-chiral proteins have shorter secondary structures and fewer internal hydrogen bonds and are less stable than homochiral proteins. Selection for hydrogen bonding yields a preponderance of L or D amino acids. Demi-chiral proteins have native global folds, including similarity to early ribosomal proteins, similar small molecule ligand binding pocket geometries, and many constellations of L-chiral amino acids with a 1.0-Å RMSD to native enzyme active sites. For a representative subset containing 550 active site geometries matching 457 (2) 4-digit (3-digit) enzyme classification (E.C.) numbers, native active site amino acids were generated at random for 472 of 550 cases. This increases to 548 of 550 cases when similar residues are allowed. The most frequently generated sequences correspond to ancient enzymatic functions, e.g., glycolysis, replication, and nucleotide biosynthesis. Surprisingly, even without selection, demi-chiral proteins possess the requisite marginal biochemical function and structure of modern proteins, but were thermodynamically less stable. If demi-chiral proteins were present, they could engage in early metabolism, which created the feedback loop for transcription and cell formation.

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Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02017-15 ◽

2015 ◽

Vol 60 (2) ◽

pp. 990-1002 ◽

Cited By ~ 7

Author(s):

Susann Skagseth ◽

Trine Josefine Carlsen ◽

Gro Elin Kjæreng Bjerga ◽

James Spencer ◽

Ørjan Samuelsen ◽

...

Keyword(s):

Active Site ◽

Active Sites ◽

Point Mutations ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

X Ray Crystallography ◽

Steady State Kinetics ◽

Hydrophilic Residues

ABSTRACTMetallo-β-lactamases (MBLs) hydrolyze virtually all β-lactam antibiotics, including penicillins, cephalosporins, and carbapenems. The worldwide emergence of antibiotic-resistant bacteria harboring MBLs poses an increasing clinical threat. The MBL German imipenemase-1 (GIM-1) possesses an active site that is narrower and more hydrophobic than the active sites of other MBLs. The GIM-1 active-site groove is shaped by the presence of the aromatic side chains of tryptophan at residue 228 and tyrosine at residue 233, positions where other MBLs harbor hydrophilic residues. To investigate the importance of these two residues, eight site-directed mutants of GIM-1, W228R/A/Y/S and Y233N/A/I/S, were generated and characterized using enzyme kinetics, thermostability assays, and determination of the MICs of representative β-lactams. The structures of selected mutants were obtained by X-ray crystallography, and their interactions with β-lactam substrates were modeledin silico. Steady-state kinetics revealed that both positions are important to GIM-1 activity but that the effects of individual mutations vary depending on the β-lactam substrate. Activity against type 1 substrates bearing electron-donating C-3/C-4 substituents (cefoxitin, meropenem) could be enhanced by mutations at position 228, whereas hydrolysis of type 2 substrates (benzylpenicillin, ampicillin, ceftazidime, imipenem) with methyl or positively charged substituents was favored by mutations at position 233. The crystal structures showed that mutations at position 228 or the Y233A variant alters the conformation of GIM-1 loop L1 rather than that of loop L3, on which the mutations are located. Taken together, these data show that point mutations at both positions 228 and 233 can influence the catalytic properties and the structure of GIM-1.

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Chromatographic method for the determination of conditional equilibrium constants for the carbamate formation reaction from amino acids and peptides in aqueous solution

Journal of the American Chemical Society ◽

10.1021/ja00069a037 ◽

1993 ◽

Vol 115 (16) ◽

pp. 7343-7350 ◽

Cited By ~ 10

Author(s):

Jian Ge Chen ◽

Mats Sandberg ◽

Stephen G. Weber

Keyword(s):

Aqueous Solution ◽

Amino Acids ◽

Chromatographic Method ◽

Equilibrium Constants ◽

Formation Reaction

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An Overview of the Proofreading Functions in Bacteria and in Severe Acute Respiratory Syndrome-Coronaviruses

International Journal of Biochemistry Research & Review ◽

10.9734/ijbcrr/2021/v30i330258 ◽

2021 ◽

pp. 33-62

Author(s):

Peramachi Palanivelu

Keyword(s):

Amino Acids ◽

Severe Acute Respiratory Syndrome ◽

Structure Function ◽

Active Site ◽

Active Sites ◽

Dna Polymerases ◽

Proton Acceptor ◽

Conserved Motifs ◽

Bacterial Dna ◽

X Ray

Aim: To understand the structure-function relationship of the proofreading (PR) functions in eubacteria and viruses with special reference to Severe Acute Respiratory Syndrome-Coronaviruses (SARS-CoVs) and propose a plausible mechanism of action for PR exonucleases of SARS-CoVs. Study Design: Bioinformatics, biochemical, site-directed mutagenesis (SDM), X-ray crystallographic data were used to study the structure-function relationships of the PR exonucleases from bacteria and CoVs. Methodology: The protein sequences of the PR exonucleases of various DNA polymerases, and RNA polymerases of SARS, SARS-related and human CoVs (HCoVs) were obtained from PUBMED and SWISS-PROT databases. The advanced version of Clustal Omega was used for protein sequence analysis. Along with the conserved motifs identified by the bioinformatics analysis, the data already available by biochemical, SDM experiments and X-ray crystallographic analysis on these enzymes were used to arrive at the possible active amino acids in the PR exonucleases of these crucial enzymes. Results: A complete analysis of the active sites of the PR exonucleases from various bacteria and CoVs were done. The multiple sequence alignment (MSA) analysis showed many conserved amino acids, small and large peptide regions among them. Based on the conserved motifs, the PR exonucleases are found to fit broadly into two superfamilies, viz. DEDD and polymerase-histidinol phosphatase (PHP) superfamilies. The bacterial DNA polymerases I and II, RNase D, RNase T and ε-subunit of DNA polymerases III belong to the DEDD superfamily. The PR enzymes from SARS, SARS-related CoVs and other HCoVs also essentially belong to the DEDD superfamily. The DEDD superfamily either uses an invariant Tyr or a His as proton acceptor during catalysis. Depending on the proton acceptor, they are further classified into DEDHD and DEDYD subfamilies. RNase T, ε-subunit of DNA polymerases III and the SARS, SARS-related CoVs and other HCoVs belong to DEDHD subfamily. However, the SARS, SARS-related CoVs and other HCoVs showed additional zinc finger motifs (ZFMs) in their active sites. DNA polymerases I, II and RNase D belong to DEDYD subfamily. The bacterial DNA polymerases X, YcdX phosphoesterases and the co-editing exonuclease of DNA polymerases III belong to the PHP superfamily. Based on the MSA, X-ray crystallographic analyses and SDM experiments, the proposed active-site proton acceptor is Tyr/His in DEDDY/H subfamilies and His in PHP superfamily of PR exonucleases. Conclusions: Based on the similarities of active site amino acids/motifs, it may be concluded that the DEDD and PHP superfamilies of PR exonucleases should have evolved from a common ancestor but diverged very long ago. The biochemical properties of these enzymes, including the four conserved acidic amino acid residues in the catalytic core, suggest that the CoVs might have acquired the exonuclease function, possibly from a prokaryote. However, the presence of two zinc fingers in the PR active site of the SARS, SARS-related CoVs and other HCoVs sets their PR exonucleases apart from other homologues.

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The Uncommon Active Site of D-Amino Acid Transaminase from Haliscomenobacter hydrossis: Biochemical and Structural Insights into the New Enzyme

Molecules ◽

10.3390/molecules26165053 ◽

2021 ◽

Vol 26 (16) ◽

pp. 5053

Author(s):

Alina K. Bakunova ◽

Alena Yu. Nikolaeva ◽

Tatiana V. Rakitina ◽

Tatiana Y. Isaikina ◽

Maria G. Khrenova ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Structural Analysis ◽

Active Site ◽

Active Sites ◽

Structural Basis ◽

Active Site Residues ◽

Type Iv ◽

Fold Type ◽

Arginine Residues

Among industrially important pyridoxal-5’-phosphate (PLP)-dependent transaminases of fold type IV D-amino acid transaminases are the least studied. However, the development of cascade enzymatic processes, including the synthesis of D-amino acids, renewed interest in their study. Here, we describe the identification, biochemical and structural characterization of a new D-amino acid transaminase from Haliscomenobacter hydrossis (Halhy). The new enzyme is strictly specific towards D-amino acids and their keto analogs; it demonstrates one of the highest rates of transamination between D-glutamate and pyruvate. We obtained the crystal structure of the Halhy in the holo form with the protonated Schiff base formed by the K143 and the PLP. Structural analysis revealed a novel set of the active site residues that differ from the key residues forming the active sites of the previously studied D-amino acids transaminases. The active site of Halhy includes three arginine residues, one of which is unique among studied transaminases. We identified critical residues for the Halhy catalytic activity and suggested functions of the arginine residues based on the comparative structural analysis, mutagenesis, and molecular modeling simulations. We suggested a strong positive charge in the O-pocket and the unshaped P-pocket as a structural code for the D-amino acid specificity among transaminases of PLP fold type IV. Characteristics of Halhy complement our knowledge of the structural basis of substrate specificity of D-amino acid transaminases and the sequence-structure-function relationships in these enzymes.

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A Novel Method for Determination of Protein in Human Serum Utilizing the Trihydroxylphenylfluorone-Molybdenum(VI) Complex as a Probe by Fluorescence Spectrometry

Journal of AOAC International ◽

10.1093/jaoac/90.1.238 ◽

2007 ◽

Vol 90 (1) ◽

pp. 238-243 ◽

Cited By ~ 1

Author(s):

Qin Wei ◽

Hongmin Ma ◽

Caihong Duan ◽

Jin Wang ◽

Shuyuan Liu ◽

...

Keyword(s):

Aqueous Solution ◽

Amino Acids ◽

Detection Limit ◽

Human Serum ◽

Fluorescence Intensity ◽

Acetate Buffer Solution ◽

Buffer Solution ◽

Protein Assay ◽

Novel Method

Abstract The fluorescence intensity of the trihydroxylphenylfluorone-molybdenum(VI) Mo(VI) complex is quenched by protein. Based on this, a novel method for protein assay in aqueous solution was developed. With pH 3.75 acetic acidsodium acetate buffer solution, in the presence of p-octyl polyethylene glycol phenyl ether microemulsion, the quenched fluorescence intensity is proportional to the concentration of bovine serum albumin (BSA) in the range of 07.00 μg/mL, and the detection limit of BSA is 5.65 ng/mL. There is no interference from amino acids and most metal ions. The method developed in this paper has been used for the successful determination of protein in human serum.

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