Human cytomegalovirus (HCMV) encodes a protease that cleaves itself and the HCMV assembly protein. Two proteolytic processing sites within the protease were identified at Ala 256-Ser 257 (release site) and Ala 643-Ser 644 (maturation site). Identification of rP5-P4' and mP4-P6' as the minimal peptide substrates spanning the release and maturation cleavage sites, respectively, demonstrated a requirement for residues flanking the conserved core in substrate recognition and hydrolysis, which are unique to HCMV. Kinetic parameters determined for release-site-derived and maturation-site-derived peptides revealed a 10-fold increase in k,,JK,, for a maturational peptide (mP4-PS') over release-site peptide (rP5-P5'). Experimental results with a panel of class-specific protease inhibitors were consistent with the protease being a member of the serine or cysteine family of proteases. Further investigation revealed that the HCMV protease activity decreased with incorporation of ['4C]iodoacetic acid, but when approximately 4.5 mol I4C were incorporatedmol enzyme, the enzyme retained approximately 20% of its original activity, indicating that hydrolysis does not require a cysteine nucleophile. Analysis of diisopropyl-fluorophosphate-inactivated protease by mass spectrometry indicated a stoichiometry of 1 diisopropyl phosphate/protease molecule, suggesting that hydrolysis requires a single serine nucleophile. The residue modified by diisopropyl fluorophosphate was identified as Ser132 by modification with 'H-labeled diisopropyl fluorophosphate, peptide mapping and Edman degradation. This residue and the region in which it is found is highly conserved among the herpes virus proteases. These data demonstrates that HCMV protease is a serine protease and that Ser132 is the active-site nucleophile.Members of the herpes virus family, including human cytomegalovirus (HCMV) and HSV-1, encode an assembly protein which is a major component of intermediate B-capsids. The assembly protein is only transiently associated with virus particles, and is absent from mature virions [l -31. As a consequence, the assembly protein is proposed to play a role in virus maturation analogous to that of the scaffolding protein of bacteriophages [4]. During infection, the HCMV preassembly protein undergoes proteolytic processing to form the assembly protein, a lower molecular-mass species that lacks 64 amino acids at the carboxy terminus [5]. Mutants of HSV-1, defective in processing the HSV-1 assembly protein, form aberrant empty capsids and fail to package DNA, indicating that proteolytic processing of the virus assembly protein is critical for herpes virus particle maturation [6, 71.The protease responsible for processing the preassembly protein during HCMV infection consists of 708 amino acids Abbreviations. HCMV, human cytomegalovirus; iPr,PF, diisopropylfluorophosphate; ESI, electrospray ionization; GST, glutathione S-transferase; HSV-1. herpes simplex virus type 1; SCMV, simian cytomegalovirus. encoded by the UL80 open reading frame, which is 3' co...

Uridine diphosphate N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) adds the first amino acid to the sugar moiety of the peptidoglycan precursor, catalyzing one of the essential steps in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Here, we report our studies on the secondary and quaternary structures of UNAM:L-Ala ligase from Escherichia coli. The molecular weight of the purified recombinant enzyme determined by electrospray ionization mass spectrometry agreed well with the molecular weight deduced from the DNA sequence. Through sedimentation equilibrium analysis, we show that the enzyme exists in equilibrium between monomeric and dimeric forms and that the dissociation constant of the dimer, Kd, was determined to be 1.1 +/- 0.4 microM at 37 degrees C and 0.58 +/- 0.30 microM at 4 degrees C. A very similar Kd value was also obtained at 37 degrees C by gel filtration chromatography. The secondary structure of the enzyme was characterized by circular dichroism spectroscopy. No change in the secondary structure was observed between the monomeric and dimeric forms of the enzyme. The activity assays at enzyme concentrations both below and above the determined Kd value lead to the conclusion that the enzyme is active both as dimers and as monomers and that the specific activity is independent of the oligomerization state.

Platelet-activating factor (PAF) was analyzed by electrospray-ionization mass spectrometry (ESI-MS) using a single quadrupole mass spectrometer. The positive-ion spectrum was dominated by an ion corresponding to a sodiated molecule when a low potential difference between the capillary exit (nozzle) and the skimmer was employed, but when the capillary exit voltage was increased, fragmentation of PAF was observed. Initial fragmentation involved the loss of the elements of trimethylamine from the sodiated molecule to yield [M+Na-59]+. An intense ion at m/z 147, generated by the loss of trimethylamine from the sodiated phosphocholine portion of the molecule was also detected, along with a lower intensity ion at m/z 184 which is representative of a protonated phosphocholine moiety. With negative-ion detection the major molecular species was [M+Cl]-. Interpretation of the mass spectral fragments was verified by ESI tandem mass spectrometry on a triple-quadrupole tandem mass spectrometer.

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