Abstract:
:Carbonic anhydrase V (CA V) is a mitochondrial enzyme that catalyzes the hydration of CO2 to produce bicarbonate and a proton. The catalytic properties of wild-type murine CA V suggest the presence of a proton shuttle residue having pKa = 9.2, the role of which is to transfer a proton from zinc-bound water to solution in the hydration direction to regenerate the zinc hydroxide form of the enzyme. Two likely candidates for shuttle residues are the tyrosines at positions 64 and 131 in the active site cavity. The crystal structure of wild-type carbonic anhydrase V [Boriack-Sjodin et al. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 10949-10953] shows that the side chain of Tyr 64 is forced into an orientation pointing away from the zinc by Phe 65, although Tyr 131 is oriented toward the zinc. We have prepared mutants of murine CA V replacing both Tyr 64 and Tyr 131 with His and Ala and investigated the proton shuttle mechanism using stopped-flow spectrophotometry and the depletion of 18O from CO2 measured by mass spectrometry. Experiments with both single and double mutations showed that neither position 64 nor position 131 was a prominent site for proton transfer. However, a double mutant of CA V containing the two replacements, Tyr 64-->His and Phe 65-->Ala, demonstrated enhanced proton transfer with an apparent pKa of 6.8 and maximal contribution to kcat of 2.2 x 10(5) s-1. In addition to the altered catalytic properties, the crystal structure of the His 64/Ala 65 double mutant strongly suggested proton transfer by His 64 after removal of the steric hindrance of Phe 65. This is the first structure-based design of an efficient proton transfer site in an enzyme.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Heck RW,Boriack-Sjodin PA,Qian M,Tu C,Christianson DW,Laipis PJ,Silverman DNdoi
10.1021/bi9608018subject
Has Abstractpub_date
1996-09-10 00:00:00pages
11605-11issue
36eissn
0006-2960issn
1520-4995pii
bi9608018journal_volume
35pub_type
杂志文章相关文献
BIOCHEMISTRY文献大全abstract::The crystal structure of a human carbonic anhydrase II (CAII) variant, cis-proline-202-->alanine (P202A), has been determined at 1.7-A resolution, indicating that the wild-type geometry, including the cis-peptidyl linkage, is retained upon substitution of proline by alanine. The CO2 hydrase activity and affinity for s...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00092a003
更新日期:1993-10-19 00:00:00
abstract::The structure of octylcarbamoyl-alpha-chymotrypsin to a resolution of 3.0 A is described. The n-octyl side chain of the active site directed irreversible inactivator octyl isocyanate is bound exclusively in the hydrophobic substrate binding pocket. The n-octyl isocyanate forms a planar urethane bond with the Ser-195 O...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00694a008
更新日期:1975-11-18 00:00:00
abstract::Cytochrome c has been chemically modified by methylene blue mediated photooxidation. It is established that the methionine residues of the protein have been specifically converted to methionine sulfoxide residues. No oxidation of any other amino acid residues or the cysteine thioether bridges of the molecule occurs du...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00650a029
更新日期:1976-03-09 00:00:00
abstract::Aspartoacylase catalyzes the deacetylation of N-acetylaspartic acid (NAA) to produce acetate and L-aspartate and is the only brain enzyme that has been shown to effectively metabolize NAA. Although the exact role of this enzymatic reaction has not yet been completely elucidated, the metabolism of NAA appears to be nec...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi052608w
更新日期:2006-05-09 00:00:00
abstract::Transported proteins often contain an extension sequence called the signal peptide. The alkaline phosphatase (PhoA) signal sequence represents a typical signal peptide for comparison to idealized sequences both in vivo and in vitro. We have designed a series of idealized signal sequences which vary in amino terminal c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00031a012
更新日期:1995-08-08 00:00:00
abstract::Several different monoclonal antibodies (mAbs) have been actively developed in the field of Alzheimer's disease (AD) for basic science and clinical applications; however, the binding kinetics of many of the mAbs with the beta-amyloid peptides (Abeta) are poorly understood. A panel of mAbs with different Abeta recognit...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi900523q
更新日期:2009-11-03 00:00:00
abstract::13C NMR has been used to demonstrate the metabolism of dilute solutions of labeled formaldehyde by Escherichia coli to methanol, formate, carbon dioxide, and several other unidentified metabolites which contain labeled CH2 groups. Aeration of bacterial suspensions within the spectrometer dramatically increased the rat...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00298a017
更新日期:1984-01-31 00:00:00
abstract::Protein carboxyl methyltransferases (EC 2.1.1.77) that catalyze the transfer of a methyl group from S-adenosylmethionine to L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely, but not universally, distributed in nature. These enzymes can participate in the repair of damaged proteins...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00092a020
更新日期:1993-10-19 00:00:00
abstract::Two sites of ligand interaction in acetylcholinesterase (AChE) were first demonstrated in ligand binding studies and later confirmed by crystallography, site-specific mutagenesis, and molecular modeling: an acylation site at the base of the active site gorge and a peripheral site at its mouth. We recently introduced a...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9813577
更新日期:1999-01-05 00:00:00
abstract::Typical cytochrome P450s secure the heme prosthetic group with a cysteine thiolate ligand bound to the iron, electrostatic interactions with the heme propionate carboxylates, and hydrophobic interactions with the heme periphery. In addition to these interactions, CYP4B1 covalently binds heme through a monoester link f...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi051267j
更新日期:2005-10-25 00:00:00
abstract::The nitrite anion is known to oxidize and degrade hemoglobin (Hb). Recent literature reports suggest a nitrite reductase activity for Hb, converting nitrite into nitric oxide. Surprisingly, no structural information about Hb-nitrite interactions has been reported. We have determined the crystal structure of the ferric...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi801015c
更新日期:2008-08-12 00:00:00
abstract::Smooth muscle cells maintain filaments of actin and myosin in the presence of ATP, although dephosphorylated myosin filaments and actin-myosin interactions are unstable under those conditions in vitro. Several proteins that stabilize myosin filaments and that stabilize actin-myosin interactions have been identified. F...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi401013g
更新日期:2013-10-29 00:00:00
abstract::Nucleotide-induced conformational changes of the catalytic beta subunits play a crucial role in the rotary mechanism of F(1)-ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated beta subunit from thermophilic Bacillus PS3 (Tbeta), the binding of this monomer to M...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi1006767
更新日期:2010-06-29 00:00:00
abstract::Hepatic microsomal NADPH-cytochrome P-450 reductase was solubilized from rabbit liver microsomes in the presence of detergents and purified to homogeneity by column chromatography. The purified reductase had a molecular weight of 78 000 and contained 1 mol each of FAD and FMN per mol of enzyme. On reduction with NADPH...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00604a032
更新日期:1978-05-30 00:00:00
abstract::The formation of pseudouridine (psi) on U5 small nuclear RNA (U5 snRNA) was studied using an in vitro modification system. Labeled U5 RNA, synthesized in vitro and therefore unmodified, was incubated in reactions containing S100 and/or nuclear extracts (NE) from HeLa cells, and the levels of psi were determined. There...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00200a025
更新日期:1994-08-30 00:00:00
abstract::The GlmU protein is a bifunctional enzyme with both acetyltransferase and uridylyltransferase (pyrophosphorylase) activities which catalyzes the transformation of glucosamine-1-P, UTP, and acetyl-CoA to UDP-N-acetylglucosamine [Mengin-Lecreulx, D., & van Heijenoort, J. (1994) J. Bacteriol. 176, 5788-5795], a fundament...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi952275a
更新日期:1996-01-16 00:00:00
abstract::S100 beta is a calcium-binding protein, which regulates the activities of several enzymes and inhibits the phosphorylation of a variety of protein kinase C substrates in a calcium-dependent manner. The protein was recently found to activate a retinal membrane guanylate cyclase, and in this paper, we report that it inh...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9802115
更新日期:1998-07-28 00:00:00
abstract::Strong absorbance of water at 1645 cm-1 has previously prevented dependable observation of infrared spectra of H2O solutions in the carbonyl region. We have obtained reliable spectra in pure water and in alcohol-water mixtures in this region and used them to study A.U polynucleotide interaction. Spectra in pure water ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00607a034
更新日期:1978-07-11 00:00:00
abstract::The effect of DNA supercoiling on a sterically very demanding threading intercalation process is investigated here. We find that the threading rate of a dimeric ruthenium complex into a negatively supercoiled plasmid at low binding density is 2 orders of magnitude higher than into the cleaved linear form. Further satu...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi802336p
更新日期:2009-02-24 00:00:00
abstract::The relationship between the domain structure of the alpha subunit of Escherichia coli tryptophan synthase and the mechanism of unfolding of the alpha subunit is investigated. Previous studies of the unfolding of the alpha subunit by increasing concentrations of guanidine hydrochloride or urea detected a partially unf...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00540a002
更新日期:1982-05-25 00:00:00
abstract::Epigallocatechin-3-gallate (EGCG) is a catechin found in green tea that can inhibit the amyloid formation of a wide variety of proteins. EGCG's ability to prevent or redirect the amyloid formation of so many proteins may reflect a common mechanism of action, and thus, greater molecular-level insight into how it exerts...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/acs.biochem.0c00043
更新日期:2020-03-17 00:00:00
abstract::The stereochemistry of proton transfer in the isomerization of [4 beta-2H]-5-androstene-3,17- dione (1d) to 4-androstene-3,17-dione (3) catalyzed by 3-oxo-delta 5-steroid isomerase (KSI) has been reinvestigated. In H2O, approximately 65% of the label is retained in the product (3); of this, one-third is at C-4 and two...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi953025x
更新日期:1996-05-21 00:00:00
abstract::Oligonucleotide-directed mutagenesis in vivo was used to create destabilizing site specific changes at position 6 in iso-1-cytochrome c in Saccharomyces cerevisiae. These changes were made in combination with the stabilizing N52I substitution. The resulting proteins showed that a variety of forces and interactions are...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00021a023
更新日期:1995-05-30 00:00:00
abstract::Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999) Biochemistry 38, 7470-7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic de...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi992537z
更新日期:2000-03-07 00:00:00
abstract::In this communication, we present data which describe optimum conditions for reverse transcription of large ribonucleic acid (RNA) templates into deoxyribonucleic acid (DNA) transcripts by the avian retrovirus reverse transcriptase in vitro. In contrast to previous studies, we have optimized all of the reaction compon...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00544a019
更新日期:1980-02-05 00:00:00
abstract::The phosphatase activity of the homodimeric NRII protein of Escherichia coli is activated by the PII protein and requires all three domains of NRII. Mutations in the N-terminal domain (L16R), central domain (A129T), C-terminal domain PII-binding site (S227R), and C-terminal domain ATP-lid (Y302N) of NRII result in dim...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi030065p
更新日期:2003-07-29 00:00:00
abstract::Structural analyses of bacterial thymidylate synthases (TSs) implicate a serine residue corresponding to Ser216 in human TS in hydrogen bond networks that are involved in binding of the nucleotide substrate, 2'-deoxyuridylate (dUMP), and that stabilize a beta-bulge in the protein. Utilizing site-directed mutagenesis, ...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi972562+
更新日期:1998-05-19 00:00:00
abstract::UDP-glucose:glycoprotein glucosyltransferase (UGT) is a soluble protein of the endoplasmic reticulum (ER) that operates as a gatekeeper for quality control by preventing transport of improperly folded glycoproteins out of the ER. We report the isolation of two cDNAs encoding human UDP-glucose:glycoprotein glucosyltran...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9916473
更新日期:2000-03-07 00:00:00
abstract::Previously, we have described significant effects of human insulin on glucose metabolism in the yeast Saccharomyces cerevisiae under conditions of growth limitation. These regulations apparently rely on a transmembrane receptor capable of binding human insulin and responding by tyrosine/serine phosphorylation of a spe...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi9920432
更新日期:2000-02-15 00:00:00
abstract::The alpha, beta, and gamma subunits of the receptor with high affinity for immunoglobulin E were isolated and their compositions assessed by direct amino acid analysis and by incorporation of radioactive precursors. The compositions show no unusual features other than a rather high content of tryptophan in the alpha c...
journal_title:Biochemistry
pub_type: 杂志文章
doi:10.1021/bi00383a024
更新日期:1987-05-05 00:00:00