High-efficiency incorporation in vivo of tyrosine analogues with altered hydroxyl acidity in place of the catalytic tyrosine-14 of Delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: effects of the modifications on isomerase kinetics.

Versions of the Y55F/Y88F modified form of Delta 5-3-ketosteroid isomerase in which the active-site tyrosine-14 is replaced by 2-fluorotyrosine, 3-fluorotyrosine, and 2,3-difluorotyrosine, amino acids having progressively greater acidity of their phenolic hydroxyls, have been expressed in an Escherichia coli host and purified to high homogeneity. The steady-state kinetic properties of Y55F/Y88F KSI and its fluorotyrosine modified forms have been determined. The mechanistic implications of the results are presented and discussed.

Insights into the catalytic mechanism and active-site environment of Comamonas testosteroni delta 5-3-ketosteroid isomerase as revealed by site-directed mutagenesis of the catalytic base aspartate-38.

Delta 5-3-Ketosteroid isomerase (KSI) of Comamonas testosteroni catalyzes the isomerization of a wide variety of delta 5(6) and delta 5(10) steroids through the formation of an enzyme bound dienol(ate) intermediate. Asp-38 has been strongly implicated in catalysis, apparently serving as a proton shuttle. In this paper the results of a detailed kinetic characterization of the KSI mutants D38E and D38H are presented. Both mutants retain significant activity, with kcat and kcat/Km values 10(3)-10(4) times greater than the D38N mutant. The results allow for a qualitative assessment of the sensitivity of the enzymes catalytic capability to the positioning and chemical nature of the catalytic base. The near identity of the ratios of kcat5-AND/kcat5,10-EST is most easily explained by a mechanism in which the second chemical step, reketonization of the intermediate dienol(ate), is not significantly rate determining. The pH dependence of the rate constants for the D38E and D38H mutants is found to be consistent with earlier proposals that an as yet unidentified titrating functional group is present in the active site and indicates that the electrostatic environment of residue 38 is hydrophobic and positively charged.

Cloning, nucleotide sequence, and overexpression of the gene coding for delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.

The structural gene coding for the delta 5-3-ketosteroid isomerase (KSI) of Pseudomonas putida biotype B has been cloned, and its entire nucleotide sequence has been determined by a dideoxynucleotide chain termination method. A 2.1-kb DNA fragment containing the ksi gene was cloned from a P. putida biotype B genomic library in lambda gt11. The open reading frame of ksi encodes 393 nucleotides, and the amino acid sequence deduced from the nucleotide sequence agrees with the directly determined amino acid sequence (K. Linden and W. F. Benisek, J. Biol. Chem. 261:6454-6460, 1986). A putative purine-rich ribosome binding site was found 8 bp upstream of the ATG start codon. Escherichia coli BL21(DE3) transformed with the pKK-KSI plasmid containing the ksi gene expressed a high level of isomerase activity when induced by isopropyl-beta-D-thiogalactopyranoside. KSI was purified to homogeneity by a simple and rapid procedure utilizing fractional precipitation and an affinity column of deoxycholate-ethylenediamine-agarose as a major chromatographic step. The molecular weight of KSI was 14,535 (calculated, 14,536) as determined by electrospray mass spectrometry. The purified KSI showed a specific activity (39,807 mumol min-1 mg-1) and a Km (60 microM) which are close to those of KSI originally obtained from P. putida biotype B.

Extent of proton transfer in the transition states of the reaction catalyzed by the delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: site-specific replacement of the active site base, aspartate 38, by the weaker base alanine-3-sulfinate.

Previous studies of the mechanism of the steroid isomerase of Comamonas (Pseudomonas) testosteroni have identified aspartate 38 as the proton porter which transfers the substrate's 4 beta proton to the 6 beta position of the product. Consequently, aspartate 38 functions as a base in the deprotonation of the substrate to form a dienol or dienolate intermediate, which then undergoes reprotonation from protonated aspartate 38 at C-6 beta to give the product. We have tried to characterize the transition states for the proton transfers by altering the pKa' of aspartate 38 and then determining the effect of the alteration on the kinetics of the enzyme. Alteration of the pKa' was accomplished by replacement of the carboxyl carbon of aspartate 38 by sulfur, a change which converts the carboxylate group to the much less basic sulfinate group. Employing Brønsted catalysis theory as applied to the individual steps of the isomerase mechanism, we find that in the enolization step of the reaction proton transfer to aspartate 38 is well advanced in the transition state. In the subsequent ketonization step, proton transfer from aspartate 38 has barely started when that transition state is reached. A series of mutant KSIs with alternative bases at position 38 have been constructed using a combination of site-directed mutagenesis and chemical modification: Asp-38 to Glu (D38E), His (D38H), and S-(carboxymethyl)cysteine (D38CMC). While the D38H and D38E mutants both retain significant isomerase activity, D38CMC is essentially inert. From the results of kinetic experiments it is possible to get a qualitative idea of the sensitivity of the enzyme's catalytic ability to the location of the base responsible for proton transfer.

Mechanism of the reaction catalyzed by delta 5-3-ketosteroid isomerase of Comamonas (Pseudomonas) testosteroni: kinetic properties of a modified enzyme in which tyrosine 14 is replaced by 3-fluorotyrosine.

Tyrosine 14 of delta 5-3-ketosteroid isomerase plays an important role in the function of the enzyme, since its replacement by phenylalanine results in a decrease in kcat by a factor of 10(-4.7). This result and the fact that this residue resides in the enzyme's substrate binding site and is in close proximity to C-2 of the bound steroid suggests that it functions as an electrophile in the catalytic mechanism by protonation of or hydrogen bonding to the C-3 carbonyl oxygen of the substrate. In order to obtain more information about the role of tyrosine 14, we have prepared a modified form of the enzyme in which tyrosine 14 has been substantially replaced in vivo by exogenously supplied 3-fluorotyrosine, a tyrosine derivative in which the pKa' of the phenol hydroxyl should be decreased by about 1.5 log units. Site specificity of this modification has been ensured by mutation of the codons for the nonessential tyrosines 55 and 88 to phenylalanine. We find that replacement of tyrosine 14 by 3-fluorotyrosine in the Y55,88F modified form of the isomerase results in a 4-fold decrease in kcat. We interpret this result in terms of a mechanism in which the transition state for enolization is dienolate-like, characterized by relatively little proton transfer from tyrosine 14 in the transition state, and the intermediate in the overall reaction is dienol-like. An alternative mechanism in which the intermediate is stabilized by a short, strong hydrogen bond can also be consistent with the data.

Allotype-dependent stimulation of peripheral blood and synovial lymphocytes by IgG3 in rheumatoid arthritis.

The immunopathologic process of rheumatoid arthritis (RA) is primarily expressed in the synovium where rheumatoid factor (RF) synthesis is concentrated. We hypothesized that RF synthesized by rheumatoid synovial cells (RSC) may be driven via a T cell-mediated immune response developed against IgG3 epitopes. To identify and characterize specific RSC RF epitopes and T cell antigens, two 28 amino acid peptides homologous with the C-terminus of IgG1 (P1) and IgG3 [G3m(5)] (P3) were synthesized and used in RF-binding studies and lymphocyte proliferation assays. Our results indicate that (i) the C-terminus of the CH3 domain contains epitopes for IgG3-reactive RSC RF; (ii) IgG3-reactive RSC RF binds primarily to IgG3 [G3m(5)]; (iii) P3 stimulated proliferation of T lymphocytes from both RA peripheral blood and RSC; and (iv) RF production was enhanced by P3 in selected RA cell cultures. These observations suggest that the C-terminus of IgG3 allotype G3m(5) may be important in T cell activation and RF production in RA.

Specific activation of a tyrosine----glycine mutant of delta 5-3-ketosteroid isomerase by phenols.

A key unknown still to be explored concerning the mechanism of delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni is the extent of the proton transfer between tyrosine-14 of the enzyme and the C-3 carbonyl oxygen of the steroid substrate. This report is a preliminary study of a system we are developing to allow us eventually to use a Brønsted analysis to measure this transfer. We describe the construction of an expression vector and tyrosine-14----glycine-14 mutant of the enzyme and its specific activation, in the manner of chemical rescue, by a variety of phenolic compounds. We suggest that the binding region of phenol is very tight and that the level of activation may be a result of steric constraints as well as of differences in the pKa' of the phenol.

Identification of serotonin from rabbit upper stomach as a stimulant of in vitro gallbladder contraction.

Using an in vitro rabbit gallbladder bioassay, the distribution and identification of bioactive substances in rabbit gastrointestinal tract were investigated. Comparison of the bioactivities of tissue extracts before and after cholecystokinin was removed by affinity chromatography demonstrated that the distributions of cholecystokinin and non-cholecystokinin substances were different. While cholecystokinin bioactivity per g of tissue was highest in the duodenum, non-cholecystokinin bioactivity was greatest in the upper stomach. The biochemical properties of the non-cholecystokinin substance in the upper stomach could not be distinguished from those of serotonin. These included molecular weights of 176, identical ultraviolet spectra, similar nuclear magnetic resonance spectra, and co-chromatography in HPLC. By weight, serotonin had 1/6th of the bioactivity of cholecystokinin octapeptide. We conclude that the principal gallbladder-contracting substance in rabbit upper stomach is serotonin.

Nucleotide sequence of the gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni.

The structural gene for the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni has been sequenced by the dideoxy method. The sequence obtained confirms the amino acid (aa) sequence of Benson et al. [J. Biol. Chem. 246 (1971) 7514-7525] at all but 5 aa residues of the 125-aa polypeptide. Amino acid residues 22, 24, 33, and 38, reported to be asparagines by Benson et al., are found to be encoded by aspartic acid codons. Amino acid residue 77, reported to be a glutamine by Benson et al., is encoded by a glutamic acid codon. The identification of aa 38 as aspartic acid, coupled with its presence in the active site, as indicated by previous affinity and photoaffinity-labeling studies and confirmed independently by x-ray crystallographic studies, strengthens the hypothesis that Asp-38 is the aa responsible for the 4 beta to 6 beta proton transfer which is part of the enzymatic reaction.

Determination of the affinity of monoclonal 19 S IgM rheumatoid factor for IgG by modified immunoassay (ELISA).

In rheumatoid arthritis (RA), the pathogenicity of IgM rheumatoid factor (RF), an autoantibody whose antigen is IgG, is still unclear although RF-IgG complexes appear to be important mediators of immune injury. The polyclonality of RF in RA makes it difficult to characterize certain qualitative properties such as specificity and affinity which may be very important in determining pathogenicity. Monoclonal IgM RF can be used to circumvent this problem. Monoclonal RF secreting cells can be produced via hybridizations with RA B lymphocytes fused with mouse or human myeloma cell lines. Another source of monoclonal RF is the sera of patients with Waldenström's macroglobulinemia (WM). One particular WM IgM RF (Kas) was chosen for our experiments to measure affinities and specificities to eight different monoclonal IgGs (three IgG1s, three IgG3s, one IgG2, and one IgG4). 19 S IgM RF, a pentavalent molecule, was mildly reduced with DTT to make 7 S univalent fragments (7 S IgM RF). 7 S IgM RF was incubated with each of the different IgGs at several different concentrations. These mixtures were allowed to come to equilibrium. An aliquot was then used to determine the amount of free 7 S IgM RF by ELISA. By plotting the reciprocal of the fraction of bound RF versus the reciprocal of the concentration of free antigen at equilibrium, different affinities were determined. The results of these determinations compare favorably with published IgM RF affinities determined by more traditional methods. This method can also be used with proteolytic digest fragments of IgG and short synthetic peptides of the IgG molecule to better locate the antigen binding site. The technique may also help us to determine whether there are select clones of RSC producing RF with different affinities that could complex to a particular type of IgG which, in vivo, could produce greater inflammatory tissue damage. Furthermore, this methodology should be useful in the study of other autoimmune diseases characterized by pathogenic autoantibodies of differing affinities.

Differential reactivity of rheumatoid synovial cells and serum rheumatoid factors to human immunoglobulin G subclasses 1 and 3 and their CH3 domains in rheumatoid arthritis.

19S IgM rheumatoid factors (RF) are polyclonal autoantibodies that may play an important pathogenic role in sustaining inflammatory synovitis in rheumatoid arthritis (RA). RF in RA have reactivity for as-yet-uncharacterized antigenic determinants in IgG Fc. We hypothesized that qualitative differences might exist between some of these RF molecules, and that differences such as reactivity and affinity might characterize more pathogenic RF molecules. Previous observations in our laboratory indicate that RF produced by rheumatoid synovial cells (RSC) have greater reactivity with human IgG and IgG3 subclass, in contrast to serum RF, which has greater reactivity with rabbit IgG and human IgG1. These observations were made using a complement-dependent RF plaque-forming cell assay. The purpose of this study was to validate and extend those observations. Therefore, we examined the reactivity of RSC and serum RF with human and rabbit IgG and the reactivity and avidity of RSC-RF for IgG1 and IgG3 molecules and Fab, F(ab')2, and pFc' fragments thereof in a solid-phase enzyme immunoassay. In particular, we found: RSC-RF had at least twice as much reactivity with human IgG as with rabbit IgG; serum RF had approximately equal reactivity with human and rabbit IgG; RSC-RF had greater reactivity and avidity for IgG3 and IgG3 pFc' than for IgG1; and RSC-RF was nonreactive with Fab or F(ab')2 from either IgG1 or IgG3. These results suggest that the major antigenic determinant for RSC-RF resides in the CH3 domain of the IgG3 molecule. Precise characterization of this epitope may provide further insight into the etiology and pathogenesis of RA.

Cloning of the gene for delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni.

We have cloned an approx. 5-kb fragment of Pseudomonas testosteroni DNA containing the structural gene of delta 5-3-ketosteroid isomerase into the EcoRI site of the lambda gt11 genome. Escherichia coli infected with these recombinant phages produce a polypeptide which is recognized by antiserum raised against the purified isomerase. Four of the recombinant lambda gt11 clones contain significant levels of isomerase activity and produce an immunopositive polypeptide of the same apparent Mr as the native isomerase obtained from P. testosteroni. The approx. 5-kb fragment hybridizes to synthetic 21-mer and 17-mer oligodeoxynucleotide mixtures corresponding to the 5' and 3' regions, respectively, of the expected nucleotide sequence of the gene.

Estimation of the relative avidity of 19S IgM rheumatoid factor secreted by rheumatoid synovial cells for human IgG subclasses.

19S IgM rheumatoid factors (RF) may play an important role in sustaining inflammation in rheumatoid arthritis (RA). As yet, no unique antigenic specificity for RF in RA has been identified. Because the synovium is central to the pathogenic changes in RA, RF produced therein might be pathogenically more important than serum RF. Therefore, we examined the reactivity and relative avidity of 19S IgM-RF in serum and rheumatoid synovial cells (RSC) from 20 patients with seropositive RA. Reactivities were determined by competitive inhibition of serum RF hemolytic activity and RSC RF-plaque-forming cells (PFC) by added soluble antigen, i.e., monomeric human IgG subclasses. Estimation of relative avidities of RSC RF for human IgG subclasses was done by calculation of fractional RF expression in the RSC RF-PFC assay following inhibition by IgG subclasses. RSC RF had greatest reactivity with IgG3 and IgG1, some reactivity with IgG2, and the least reactivity with IgG4. Serum RF reacted most with IgG1 and IgG2, reacted some with IgG4, but reacted poorly with IgG3. The antigenic determinants with which RSC RF reacted were common to many IgG3 molecules. The highest relative avidity of RSC RF was for IgG3. These observations indicate a selective deficiency of serum RF compared with RSC RF and suggest an important pathogenic role for these qualitatively different RSC RF molecules for in situ RF immune complex-mediated inflammation in RA synovial tissue.

The amino acid sequence of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B.

We have determined the primary structure of a delta 5-3-oxosteroid isomerase from Pseudomonas putida biotype B. The enzyme is a dimeric protein of two identical subunits, each consisting of a polypeptide chain of 131 residues and a Mr = 14,536. The intact S-carboxymethyl protein was sequenced from the NH2 terminus using standard automated Edman degradation and automated Edman degradation using fluorescamine treatment at known prolines to suppress background. The isomerase was fragmented using CNBr, trypsin, iodosobenzoic acid, and acid cleavage at aspartyl-prolyl peptide bonds. The peptides resulting from each fragmentation were separated by reversed-phase high performance liquid chromatography and sequenced by automated Edman degradation. The full sequence was deduced by the overlapping of the various peptides. A search for homologous proteins was performed. Only the oxosteroid isomerase from Pseudomonas testosteroni, an expected homology, was found to be similar. Comparison of the two proteins shows that the region of strongest homology is the region containing the aspartic acid at which steroidal affinity and photoaffinity reagents have been shown to react in the P. testosteroni isomerase. The P. putida isomerase contains 3 cysteines and 2 tryptophans, whereas the P. testosteroni isomerase lacks these amino acids. The two proteins are not highly conserved.

Use of a solid-phase photoaffinity reagent to label a steroid binding site: application to the delta 5-3-ketosteroid isomerase of Pseudomonas testosteroni.

In order to extend our analysis of the reactions that occur during the active site directed photoinactivation of delta 5-3-ketosteroid isomerase sensitized by unsaturated steroid ketone photoaffinity reagents, the site of covalent attachment has been identified. A solid-phase photoaffinity reagent, delta 6-testosterone-agarose, has been employed for this purpose; this type of reagent, in contrast to solution-phase reagents, facilitated the recovery of a peptide fragment of the isomerase bearing the residue at which covalent attachment had occurred. Amino acid analysis and sequence determination of the peptide provided evidence that the site of attachment was aspartate-38. This result, in combination with the low-resolution crystallographic structure of the enzyme [Westbrook, E. M., Piro, O. E., & Sigler, P. B. (1984) J. Biol. Chem. 259, 9096-9103], suggests that aspartate-38 is located in the vicinity of the bottom of the steroid-binding pit. The potential usefulness of solid-phase photoaffinity reagents in the identification of sites of covalent attachment on target proteins such as hormone receptors is discussed.

An inexpensive computerized enzyme kinetics system based on a Gilford spectrophotometer and an Apple IIe microcomputer.

A microcomputer-controlled data acquisition system for spectrophotometric enzyme kinetics measurements has been assembled. The system uses an Apple IIe computer which is interfaced to the binary coded decimal output of a Gilford spectrophotometer. No analog-to-digital converter had to be purchased. A BASIC program which collects timed absorbance readings every 500 ms, plots the data in real time, performs a linear regression of the data to measure the reaction rate, and calculates the enzyme activity concentration is given in full. Details describing the interfacing of the computer to the spectrophotometer are presented which will permit other laboratories to readily assemble their own systems using this hardware. Kinetic data acquired by the system are highly reproducible and agree well with data processed much more slowly by manual techniques from strip chart recordings.

Photoaffinity modification of delta 5-3-ketosteroid isomerase by light-activatable steroid ketones covalently coupled to agarose beads.

In order to identify the minor site(s) of photoattachment of unsaturated steroid ketones to delta 5-3-ketosteroid isomerase from Pseudomonas testosteroni, we have developed a solid-state photoaffinity labeling technique. Two solid-state reagents, O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-19-nortestosterone and O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadien-3-one, have been synthesized. Under anaerobic conditions, isomerase bound to these resins is photoinactivated by UV light (lambda greater than 290 nm) whereas isomerase bound to O-carboxymethylagarose-ethylenediamine-deoxycholate or isomerase in the presence of O-carboxymethylagarose-ethylenediamine-acetate is almost completely stable to irradiation under the same conditions. Photoinactivation under anaerobic condition promoted by the resin-bound steroid ketones results from a reaction at the active site since the competitive inhibitor, sodium cholate, which does not absorb light above 290 nm, provides protection toward photoinactivation. Preliminary analysis of isomerase that has been photolyzed in the presence of O-carboxymethylagarose-ethylenediamine-succinyl-17 beta-O-4,6-androstadiene-3-one has established that the enzyme is converted to at least two different forms. One form binds more tightly to the resin than does the native enzyme. This form can be eluted by a sodium dodecyl sulfate containing buffer. The second form is not eluted by this buffer but can be released from the resin by cleavage of the ester bond linking the steroid to the derivatized agarose. We presume that the latter form is covalently coupled to the resin-linked steroid. In the presence of oxygen, additional nonspecific inactivation reactions occur, but these can be suppressed by the singlet oxygen trap, L-histidine. The application of solid-state photoaffinity reagents to some areas of receptor isolation and characterization is discussed.

The purification and characterization of delta 5-3-ketosteroid isomerase from Pseudomonas putida, a cysteine-containing isomerase.

A delta 5-3-ketosteroid isomerase (EC 5.3.3.1) has been isolated from Pseudomonas putida Biotype B and purified to homogeneity. This previously undescribed steroid isomerase resembles that isolated from Pseudomonas testosteroni (Talalay, P., and Wang, V.S. (1955) Biochim. Biophys. Acta 18, 300-301). The enzyme is induced by various steroids, has a subunit molecular weight of 13,750 +/- 250, a pI of 4.8 +/- 0.1 has a specific activity of 40,000 units/mg, using 5-androstene-3,17-dione as the substrate. The amino acid composition of the enzyme subunit is Lys 2, His 2, Arg 8, Asp 11, Thr 5, Ser 4, Glu 17, Pro 8, Gly 12, Ala 12, Val 10, Met 5, Ile 6, Leu 8, Tyr 4, Phe 4, and Cys 4. The amino acid sequence has been determined for the NH2-terminal 50 residues. This portion of the polypeptide chain is approximately 47% homologous with the amino acid sequence of the first 50 residues of the delta 5-3-ketosteroid isomerase from P. testosteroni. The amino acid sequence of residues 33 to 41 of the P. putida isomerase is identical with the region Ala 31 through Pro 39 in the P. testosteroni enzyme. Residue 40 of the P. putida enzyme is aspartic acid and corresponds in the sequence to Asp 38 of the P. testosteroni isomerase, which has been shown to be essential for enzymatic activity (Ogez, J.R., Tivol, W.F., and Benisek, W.F. (1977) J. Biol. Chem. 252, 6151-6155).