Daniel H. Hug

Light activation of enzymes.

This review encompasses new developments in the activation of enzymes by light and is based on literature available from 1977 to mid 1980 to provide continuity with the last Yearly Review on this subject [98]. We have adopted a broad definition for enzyme photoactivation. Therefore, this subject overlaps some of the Yearly Reviews since 1977 to which we have referred the reader when appropriate. The synthesis of carotenoids induced by near-UV light in microorganisms was covered in a Yearly Review [38]. Specialized reviews are noted under each topic. General reviews have been prepared by Erlanger [36], Montagnoli [98] and Hug [49].

Sulfhydryl groups in urocanase: Relationship to nicotinamide adenine dinucleotide binding☆

Abstract This study concerned the role of the sulfhydryl groups in urocanase of Pseudomonas putida . When p -chloromercuribenzoate was added to the enzyme, two sulfhydryl groups reacted at once with little inhibition; the enzyme slowly became inhibited while further sulfhydryls reacted. After the p -chloromercuribenzoate inhibition occurred, if a thiol was subsequently added, most of the original activity was recovered. As the incubation time with p -chloromercuribenzoate was increased, the thiol became less effective in reversing the inhibition. However, if NAD + (10 μ m ) was added with the thiol, 60–90% of the initial activity was restored even after long p -chloromercuribenzoate incubations. Restoration of activity by NAD + was concentration dependent and specific for NAD + . Radioactive NAD + could be bound to urocanase. These results confirm the coenzyme role for NAD + in urocanase. In urea, p -chloromercuribenzoate titration of urocanase measured 11.9 -SH groups per molecule. Sulfite-modified enzyme treated with p -chloromercuribenzoate and dialyzed was substantially photoactivated in the presence of a thiol; that is, NAD + was not required to restore activity. From these results, it is proposed that this enzyme contains two reactive —SH groups and that an essential —SH group is involved in NAD + binding. Forces present in the sulfite-modified enzyme prevent the release of the NAD + in the presence of mercurials.

Photomodulation of Enzymes

The photomodulation of enzymes involves the activation and inactivation of enzyme reactions by UV and visible light. Enzymes or their reactions may be affected directly or indirectly. Direct effects involve photoproduction of a substrate, photodissociation of an inhibitor, photochemistry of protein amino acids, irradiation of a chromophore and irradiation of an enzyme substrate. Indirect effects involve gene expression, phytochrome and other photoreceptors which are not part of the enzyme, protein synthesis, membranes and photosynthesis. Photoactivation of enzymes is related to photocarcinogenesis, photomorphogenesis of plants, primary effects or side effects of phototherapy, deoxyribose nucleic acid (DNA) repair and many other aspects of biology and medicine. Model systems may contribute to the knowledge of protein chemistry and medicinal chemistry.Light is an important environmental signal. An organism survives by its ability to adapt to the conditions of its environment; many organisms are known to respond to light and photoresponsive molecules are widespread in nature. The interaction of light with an enzyme is one of the possible means for biological systems to respond. Light may influence either an increase or decrease in enzyme activity; thus light affects the chemistry of the cell. These processes can be very complex (e.g., photomodulation of chloroplast enzymes) or they can be remarkably simple (e.g., photoactivation of urocanase). Reversion is an essential part of photomodulation.

New trends in photobiology: Photomodulation of enzymes

Abstract The photomodulation of enzymes involves the activation and inactivation of enzyme reactions by UV and visible light. Enzymes or their reactions may be affected directly or indirectly. Direct effects involve photoproduction of a substrate, photodissociation of an inhibitor, photochemistry of protein amino acids, irradiation of a chromophore and irradiation of an enzyme substrate. Indirect effects involve gene expression, phytochrome and other photoreceptors which are not part of the enzyme, protein synthesis, membranes and photosynthesis. Photoactivation of enzymes is related to photocarcinogenesis, photomorphogenesis of plants, primary effects or side effects of phototherapy, deoxyribose nucleic acid (DNA) repair and many other aspects of biology and medicine. Model systems may contribute to the knowledge of protein chemistry and medicinal chemistry.

IN VIVO ROLE OF SULFITE IN PHOTOCONTROL OF UROCANASE FROM Pseudomonas putida

Abstract— Urocanase from Pseudomonas putida becomes inactive in growing and resting cells and is activated by UV radiation. Sulfite addition to the bound nicotinamide adenine dinucleotide coenzyme has previously been shown to inactivate the enzyme in vitro. The enzyme released sulfite upon photoactivation. Whether sulfite addition and dissociation is involved in the in vivo photoregulation of urocanase was examined in this study. The dark reversion (inactivation) in cultures was markedly enhanced by growth at 32°C rather than at 24°C; cells grown at 32°C and resting cells were used to obtain in Wvo‐inactivated urocanase. We purified the in vivo‐inactivated enzyme and quantitated the amount of sulfite released through photodissociation. One mole of sulfite was released per mole of urocanase. This is based on a molecular weight of 110000 confirmed by gel electrophoresis and a protein estimation method validated by our data. Our previous report of sulfide inactivation of urocanase in vitro is now shown to have been mistaken; the inactivation resulted from the oxidation of sulfide to sulfite, which occurred in solution.

TRYPTOPHANYL FLUORESCENCE QUENCHING OF UROCANASE FROM Pseudomonas putida BY ACRYLAMIDE, CESIUM, IODIDE, and IMIDAZOLEPROPIONATE

Abstract— Urocanase from Pseudomonas putida can be photoactivated by UV radiation. Because of the action spectrum peak at 280 nm, tryptophan has been implicated in the photoactivation by energy transfer. In these studies, tryptophan was determined, the exposure and environment of tryptophanyl residues were studied with collisional quenchers, and the involvement of tryptophanyl residues in the photoactivation of urocanase was investigated. There are sixteen tryptophanyl residues per urocanase molecule as measured by two methods. Fluorescence quenching with acrylamide, cesium, and iodide was used to describe the accessibility and environment of urocanase tryptophanyl residues. Quenching constants indicated there is little difference in the accessibility of tryptophanyl residues between active and inactive enzyme. Tryptophanyl residues of native enzyme were most accessible to acrylamide (fa, = 0.6–0.7), less accessible to iodide (fa= 0.4), and not accessible to cesium ion, suggesting that surface residues were in regions of positive charge. Stern‐Volmer plots indicated a heterogeneous population of tryptophanyl residues with different accessibilities. A competitive inhibitor, imidazolepropionate, quenched fluorescence; the quenching was used to determine the dissociation constant for the enzyme‐inhibitor complex (Kd= 0.20 mM). Kinetic data showed Ki= 0.25 mM. Mixed quencher experiments indicated that the tryptophanyl residues quenched by imidazolepropionate were more accessible to acrylamide and less accessible to iodide. These studies suggest that the residues involved in putative energy transfer during photoactivation are not fully exposed.

Photoactivation of Urocanase in Pseudomonas putida; Activation by a Biochemically Generated Excited Statea

In recent years there have been numerous published reports of the generation of electronically excited states in dark biochemical systems. It has been proposed that, through these excited states, the cell may be able to promote photochemical processes in the absence of light.’ One of the systems that has been investigated is the horseradish peroxidase (HRP) catalyzed aerobic oxidation of indole-3-acetic acid (IAA), which generates indole-3-aldehyde (IAI) in the excited triplet state.2 This chemi-energized species is capable of transferring energy to appropriate acceptors and thus promoting photochemical processes. Among these are thiouridine phosphorescence in transfer RNA: DNA strand scission through a singlet oxygen intermediate: and red emission from chloroplasts.’ We became interested in whether we could elicit a “photoactivation without light” of urocanase from Pseudomonas putida. Urocanase, the second enzyme of histidine catabolism, was shown to be photoactivated by UV light in a report from this laboratory! The activation consists of a photodissociation of sulfite from the tightly bound NAD+ coenzyme.’ The unblocked NAD’ moeity can then function in the enzyme reaction? The photocontrol of the histidine catabolic pathway through photoactivation of urocanase has, therefore, already been demonstrated in P. purida. We now report that the activation of urocanase can be induced in the absence of light when the enzyme is exposed to the IAA/HRP/02 system. When urocanase, purified from P. putida, is incubated in the dark with the IAA/HRP/02 system at pH 5.5 and 2 Y C , the enzyme is clearly activated as compared to a control, minus H R P (FIGURE 1). The activation is produced through a product of the peroxidase-catalyzed oxidation of the IAA substrate, because no increase in urocanase activity was observed for a minus IAA control. Further, the extent of activation after a 5 hr incubation is equal to the maximal activation achievable by near UV radiation under these experimental conditions. The photochemical-like activation in the dark is most likely produced by a direct energy transfer from the triplet donor (IA1). The involvement of a singlet oxygen intermediate is excluded by the lack of effect of the singlet oxygen quencher, methionine (TABLE 1). Likewise, superoxide ion probably does not participate in the activation as evidenced by the lack of effect of superoxide dismutase; the involvement of the hydroxyl radical is ruled out because sodium benzoate does not prevent activation. Efforts are presently underway to further elucidate the energy transfer leading to activation.

EFFECT OF TEMPERATURE ON SULFITE-MEDIATED DARK REVERSION OF UROCANASE IN Pseudomonas putida

Abstract. Cultures of Pseudomonas putida produce inactive but photoactivatable urocanase. The ratio of active to inactive urocanase is much lower in cells grown above 30°C than below 30°C. Inactivation results from the addition of sulfite to the tightly but noncovalently bound nicotinamide adenine dinucleotide. Ultraviolet light restores activity by dissociation of the adduct. The Arrhenius plot of the inactivation of purified urocanase by sulfite appeared linear from 22 to 38°C. Urocanase does not have an increased vulnerability to sulfite above 30°C because of a change in the rate‐limiting step or a conformational change of the protein. Cell extracts and growth medium were analyzed for sulfite and sulfite was confirmed by susceptibility to sulfite oxidase. As the growth temperature was raised from 24 to 32°C, intracellular sulfite rose from 270 μM to 400 μM and the ratio of active to inactive urocanase dropped from 11.8 to 1.9. Above 32°C the intracellular sulfite concentration decreased, the sulfite concentration of the growth medium rose sharply, and the ratio of active to inactive urocanase activity fell further. P.‐putida became leaky, at least to sulfite, above 32°C. We conclude that sulfite formation or degradation is altered to produce more sulfite in cultures grown at elevated temperatures resulting in reversion of urocanase to a photoactivatable form.

Evidence against a temperature-dependent conformational change in urocanase from Pseudomonas putida

Enzymatic activity of urocanase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) has an unusual resistance to temperature changes, and a temperature-dependent conformational change has been suggested (Hug, D.H. and Hunter, J.K. (1974) Biochemistry 13, 1427-1431). A conformational change or dissociation has been proposed in the range of 29-31 degrees C (Cohn, M.S., Lynch, M.C. and Phillips, A.T. (1975) Biochim. Biophys. Acta 377, 444-453). In this work, no evidence was found for a temperature-dependent conformational change or dissociation. Arrhenius plots of Km and Vmax were linear; the sedimentation coefficient was independent of temperature; tryptophanyl fluorescence was a linear function of temperature; and heat capacity calorimetry showed no transitions below 60 degrees C.

Thioglycolate, competitive inhibitor of urocanase

Abstract Urocanase was inhibited by thioglycolate, 2-mercaptoethanol, dithioerythritol, and 3-mercaptopropionate. Thioglycolate inhibited competitively at low concentrations (Ki, 0.1 mM) and protected the active site from modification by sulfite. The inhibited enzyme was reactivated by dialysis. A difference spectrum peak of 328 nm for the thioglycolate-urocanase complex compared to the 327 nm absorption maximum of the NAD-thioglycolate adduct. Several nucleophiles are known to inhibit urocanase. We conclude that thioglycolate, as a nucleophilic agent, inhibits by forming an adduct with the tightly bound NAD of urocanase. These results provide indirect evidence that NAD may be the locus of substrate binding in urocanase.